CN117467801A - Products and methods for rapid identification of Yersinia pestis-specific phages - Google Patents

Abstract

The present invention relates to products and methods for rapid identification of Yersinia pestis-specific phages. Specifically, the present invention relates to nucleic acid detection products and isolation and culture methods for Yersinia pestis-specific phages in biological specimens. The products and methods can be used to quickly and accurately identify Yersinia pestis-specific phages.

Description

Products and methods for rapid identification of Yersinia pestis-specific phages

Technical field

The present invention relates to products and methods for rapid identification of Yersinia pestis-specific phages. Specifically, the present invention relates to nucleic acid detection products and isolation and culture methods for Yersinia pestis-specific phages in biological specimens. The products and methods can be used to quickly and accurately identify Yersinia pestis-specific phages, and by This supports the existence of Yersinia pestis.

Background technique

Plague is a severe infectious disease caused by Yersinia pestis. Humans are infected through the bite of infected fleas, contact with infected animals, or inhalation. If not treated in time, the fatality rate is as high as 30% to 100%. According to the "Law of the People's Republic of China on the Prevention and Control of Infectious Diseases", plague is a Class A infectious disease in my country. It is highly contagious and has a high mortality rate. There are mandatory requirements for epidemic reporting and review time limits. Therefore, the rapid detection of Yersinia pestis is of great practical significance for the timely diagnosis and treatment of plague patients and epidemic management, and is also of great significance for ensuring the normal operation of social production and life.

According to B.6.1 of the "Plague Diagnostic Standards/WS279-2008", the key to identifying Y. pestis is its ability to be lysed by Y. pestis phage. Additionally, when Y. pestis is difficult to isolate from lesions or decayed material, the isolation of Y. pestis phages is strong evidence that Y. pestis may be present. Therefore, the isolation of Y. pestis phages from biological specimens can, on the one hand, prove the existence of Y. pestis, and on the other hand, the isolated Y. pestis phages are expected to be used for the identification of Y. pestis. .

The existing isolation strategy for Y. pestis phage is to first use Y. pestis to expand the phage propagation culture of the target sample, then lyse the cells with chloroform, and use the top agar phage sensitivity test to detect the presence of phages. However, this strategy has the following limitations: First, the test cycle is long and requires a lot of manpower and material resources. Secondly, while chloroform lyses bacterial cells, it also causes damage to phages, and the sample volume tested by the top agar method is limited. Therefore, this strategy has higher requirements for the detection limit of phage and lower sensitivity. A more serious problem is that the phages isolated by this strategy may not be specific for Y. pestis.

In addition, when there are a large number of samples to be tested and the conditions of primary laboratories are limited, rapid and accurate early detection methods are needed to screen out non-target samples, thereby reducing the number of target samples that need to be cultured in the later stage and reducing the burden and burden on operators. Later occupation of laboratory resources.

Therefore, there is a need in this field for a method for rapid identification of Yersinia pestis-specific phages in biological specimens. It must have the advantages of high sensitivity and strong specificity, be suitable for processing larger batches of samples, and be able to quickly and accurately identify Provide identification results.

Contents of the invention

The present invention establishes a method for detecting, isolating and cultivating specific bacteriophage of Yersinia pestis from biological specimens. The method adopts the strategy of first conducting PCR detection on the sample nucleic acid, confirming the positive, and then directional isolation and culturing. Specifically, biological specimens containing Y. pestis-specific phages are identified through nucleic acid detection, and then Y. pestis is used to propagate the phages of the positive specimens, and finally their lysis phenotype is verified. The advantages of the method of the present invention are: firstly, the nucleic acid detection in the first step has high sensitivity and can detect target samples with low phage content. The subsequent phage multiplication of the target sample can further increase the phage content in the sample and avoid problems due to low phage content. resulting in false negatives for phage lysis. Secondly, field tissue samples collected as epidemiological samples are susceptible to interference from background nucleic acid substances in the host animal and the wild environment. Therefore, specific detection methods are needed to avoid false positives and severe background noise. However, in this complex background environment, it is impossible to predict in advance which primers can meet this requirement. After repeated verification with real biological samples, the inventors were surprised to find that the specific primers provided by the present invention can avoid the interference of host animals and background nucleic acid substances in the wild environment, exhibit good specificity and signal-to-noise ratio, and avoid Eliminate false positives and interference from background nucleic acid substances. Third, the target sample is locked through the first step of nucleic acid detection, eliminating the need for phage proliferation and phenotypic experiments on other non-target samples, which can significantly improve the efficiency of phage isolation in the sample. Therefore, the method of the present invention can significantly reduce unnecessary sample phage proliferation work from the source, thereby saving valuable laboratory resources and reducing the workload of experimental operators. In addition, the bacterial removal step of the method of the present invention does not involve chloroform, and instead uses a 0.22 μm pore size filter for sterilization, which can retain the original state of the phage in the biological sample to the greatest extent, and is more conducive to ensuring the reliability of the results.

The method of the present invention first eliminates non-Yersinia pestis-specific phages through sample nucleic acid detection. The fragments detected are the Yersinia pestis diagnostic phage Yep-phi and the Yersinia pestis phage YepMm independently isolated by the inventor of the present invention (A Lytic Yersina pestis Bacteriophage Obtained From the BoneMarrow of Marmota himalayana in a Plague-Focus Area in China, Frontiers in Cellular and Infection Microbiology, 08Jul 2021,11:700322) Specific nucleic acid fragments of these two phages. The genome nucleotide identity of YepMm and Yep-phi is 99.99%, and both have high specificity for lysing Yersinia pestis. YepMm and Yep-phi both belong to the Yep-phi group, but the nucleotide identity between YepMm and other phages in this group is less than 97.91%, and other phages in this group also have lytic effects on other strains such as Shigella sonnei (Bacteriophage .2012Jul 1;2(3):168–177), therefore, the specific fragments of Yep-phi and YepMm were selected for nucleic acid detection, which has higher specificity for identifying Yersinia pestis-specific phages.

Therefore, the present invention provides new Yersinia pestis-specific phage nucleic acid detection products and detection and isolation and culture methods, which avoid many shortcomings such as low sensitivity and poor specificity of previous plague phage isolation and identification methods, and have high sensitivity. It has the advantage of strong specificity and can process large batches of samples, provide results quickly and accurately, and reduce the workload of experimental operators.

1. Primers

In a first aspect, the present invention provides a pair of nucleic acid primers for identifying Yersinia pestis-specific phages in biological specimens, wherein the target nucleic acid sequence amplified by the primers is the 19th, 197th to 197th to 197th of SEQ ID No. 1. A sequence between 19,933 nucleotides (SEQ ID No. 2) or any fragment thereof, wherein the length of the fragment is not less than 100 bp.

In one embodiment, the length of the target nucleic acid sequence amplified by the primer or any fragment thereof is 100bp to 737bp, preferably the length is 200bp to 737bp, 300bp to 737bp, 400bp to 737bp, 500bp to 737bp, 600bp to 737bp, or 700bp to 737bp, e.g. length 100, 150, 200, 250, 300, 350, 400, 450, 500, 510, 550, 600, 650, 700bp or 737bp.

In one embodiment, the primers are each independently 10 bp to 50 bp in length, such as 15 bp to 50 bp, such as 15 bp to 40 bp, 15 bp to 35 bp, 15 bp to 30 bp, 15 bp to 25 bp, or 18 bp to 23 bp, such as 15, 16 , 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30bp.

In one embodiment, the present invention provides a pair of nucleic acid primers for identifying Yersinia pestis-specific phages in biological specimens, wherein the target nucleic acid sequence amplified by the primers is the 19,380th position of SEQ ID No. 1 to the 19,889th nucleotide (SEQ ID No. 3) or any fragment thereof, wherein the length of the fragment is not less than 100 bp.

In one embodiment, the length of the target nucleic acid sequence amplified by the primer or any fragment thereof is 100 bp to 510 bp, preferably the length is 200 bp to 510 bp, 300 bp to 510 bp, 400 bp to 510 bp, or 500 bp to 510 bp, for example, the length is 100 bp , 150, 200, 250, 300, 350, 400, 450, 500 or 510bp.

In one embodiment, the primers are each independently 10 bp to 50 bp in length, such as 15 bp to 50 bp, such as 15 bp to 40 bp, 15 bp to 35 bp, 15 bp to 30 bp, 15 bp to 25 bp, or 18 bp to 23 bp, such as 15, 16 , 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30bp.

In another embodiment, the present invention provides a pair of nucleic acid primers for identifying Yersinia pestis-specific phages in biological specimens, wherein the forward primer corresponds to No. 19,197 to No. 19,379 of SEQ ID No. 1 The sequence between nucleotides (SEQ ID No. 4) or any fragment thereof, and the reverse primer corresponds to the sequence between 19,890th and 19,933rd nucleotides of SEQ ID No. 1 (SEQ ID No. 1). 5) or any fragment thereof. In a preferred embodiment, the lengths of the forward primer and the reverse primer are each independently 10 bp to 50 bp, such as 15 bp to 50 bp, such as 15 bp to 40 bp, 15 bp to 35 bp, 15 bp to 30 bp, 15 bp to 25 bp, or 18bp to 23bp, such as 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30bp.

In one embodiment, the primer is in the form of a nucleic acid, such as DNA. Alternatively, the primer may be in the form of a nucleic acid analog, such as PNA (peptide nucleic acid), LNA (locked nucleic acid) or BNA (bridged nucleic acid). The primer may also be a nucleic acid that has been partially replaced by LNA or PNA.

In a preferred embodiment, the primer sequence is selected from:

F da:5’-ATgTCCggTgAATCTAATgCTg-3’

R da: 5’-CCAAAAgTCTTAgTACggCTTgC-3’

where F and R represent forward and reverse primers respectively; or

F xiao：5’-ggTgAAgAAgAAgACgAgAAT-3’

R xiao：5’-CTTAACggTCgCTAggTC-3’

where F and R represent forward and reverse primers respectively.

In one embodiment, the invention provides a combination of two pairs of nucleic acid primers for identifying Yersinia pestis-specific phages in biological specimens, including:

(1) A first pair of primers, wherein the forward primer corresponds to the sequence between 19,197th and 19,379th nucleotide of SEQ ID No. 1 (SEQ ID No. 4) or any fragment thereof, and the reverse primer A complementary sequence corresponding to the sequence between 19,890th and 19,933rd nucleotides of SEQ ID No. 1 (SEQ ID No. 5) or any fragment thereof, wherein the forward primer and the reverse primer have respective lengths Independently 10bp to 50bp, such as 15bp to 50bp, such as 15bp to 40bp, 15bp to 35bp, 15bp to 30bp, 15bp to 25bp, or 18bp to 23bp, such as 15, 16, 17, 18, 19, 20, 21, 22 , 23, 24, 25, 26, 27, 28, 29 or 30bp; and

(2) A second pair of primers, wherein the target nucleic acid sequence amplified by the primers is the sequence between the 19,380th and 19,889th nucleotides of SEQ ID No. 1 (SEQ ID No. 3) or any fragment thereof , wherein the length of the fragment is not less than 100bp, and the length of the primers is each independently 10bp to 50bp, such as 15bp to 50bp, such as 15bp to 40bp, 15bp to 35bp, 15bp to 30bp, 15bp to 25bp, or 18bp to 23bp, such as 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30bp.

In a preferred embodiment, the length of the target nucleic acid sequence amplified by the second pair of primers or any fragment thereof is 100 bp to 510 bp, preferably 200 bp to 510 bp, 300 bp to 510 bp, 400 bp to 510 bp, or 500 bp to 510 bp. , such as 100, 150, 200, 250, 300, 350, 400, 450, 500 or 510bp in length.

In a preferred embodiment, the sequence of the first pair of primers is:

F da:5’-ATgTCCggTgAATCTAATgCTg-3’

R da: 5’-CCAAAAgTCTTAgTACggCTTgC-3’

where F and R represent forward and reverse primers respectively.

In a preferred embodiment, the sequence of the second pair of primers is:

F xiao：5’-ggTgAAgAAgAAgACgAgAAT-3’

R xiao：5’-CTTAACggTCgCTAggTC-3’

where F and R represent forward and reverse primers respectively.

In a preferred embodiment, the present invention provides a combination of two pairs of nucleic acid primers for identifying Yersinia pestis-specific phages in biological specimens, including:

(1) The first pair of primers, which are

F da:5’-ATgTCCggTgAATCTAATgCTg-3’

R da: 5’-CCAAAAgTCTTAgTACggCTTgC-3’

where F and R represent forward and reverse primers respectively; and

(2) The second pair of primers, which are

F xiao：5’-ggTgAAgAAgAAgACgAgAAT-3’

R xiao：5’-CTTAACggTCgCTAggTC-3’

where F and R represent forward and reverse primers respectively.

In a preferred embodiment, the Y. pestis specific phage is YepMm phage and/or Yep-phi phage.

2. Phage detection kit

In a second aspect, the present invention provides a detection kit for identifying Yersinia pestis-specific phage in biological specimens, which includes the nucleic acid primer as described in the first aspect.

In one embodiment, the present invention provides a detection kit for identifying Yersinia pestis-specific phages in biological specimens, which includes the nucleic acid primers described in the first aspect and instructions for use.

In one embodiment, the present invention provides a detection kit for identifying Yersinia pestis-specific phages in biological specimens, which includes the nucleic acid primers described in the first aspect, instructions for use and/or for Reagents for PCR reactions, such as polymerase, purified water, buffer, dNTP mixture, and optional positive controls.

In a preferred embodiment, the reagents used in the PCR reaction include Taq DNA polymerase, Taq buffer (Mg ²⁺ solution) and dNTP mixture.

In a preferred embodiment, the detection kit of the present invention includes multiple containers, such as Eppendorf tubes, bags, boxes, vials or vials, each of which contains the following reagents: commercial Premix (containing DNA Polymerase, Buffer, dNTP Mixture), ultrapure water, and one or two pairs of primers mentioned above. For example, commercial Premix can contain Taq DNA Polymerase 1.25U, Taq Buffer (3mM Mg ²⁺ ), and dNTPMixture 0.4mM each.

In a preferred embodiment, the detection kit of the present invention contains one or more reagents in the following PCR reaction system:

(1)DNA template 1μl

(2) Forward primer 0.5μl (10μm)

(3) Reverse primer 0.5μl (10μm)

(4)ddH ₂ O 8μl

(5) Premix 10μl, its ingredients are as follows:

Taq DNA Polymerase 1.25U

Taq Buffer 3mM Mg ²⁺

dNTP Mixture 0.4mM each.

It should be understood that any technical features, embodiments, preferred embodiments and any combination thereof of the primers described in the first aspect above can be introduced into the second aspect, resulting in all embodiments of the detection kit of the second aspect. All are included in the scope of the present invention.

The instructions for use may include information on one or more of the following instructions: how to use the nucleic acid primers described in the first aspect to perform a PCR reaction to detect the presence of Yersinia pestis-specific phages in biological specimens, the organism to be tested Sample processing methods, as well as PCR reaction conditions and reagents, etc.

The instruction manual may be a paper, insert, booklet, digital storage medium, optical storage medium, audio presentation or visual display material containing the information and instructions. In some embodiments, the instructions for use can be browsed or downloaded from a website containing a display of the information and instructions, or can be visual display materials, promotional materials, product labels, package inserts, advertisements, handouts, announcements, Audio tape, video tape, DVD, CD-ROM, computer-readable chip, computer-readable card, computer-readable disk, computer memory, USB device, FireWire device, or any combination thereof.

In one embodiment, the detection kit of the invention is in the form of a single package or a virtual package.

The term "single package" means that the components of a kit are physically combined in or with one or more containers and considered a unit of manufacture, distribution, sale, or use. Such containers include, but are not limited to, Eppendorf tubes, bags, boxes, cartons, bottles, packaging of any type or design or material, overwraps, shrink wraps, attached components (eg, stapled or adhesive), or combinations thereof. A unitary package may be one or more containers containing the components of a kit and instructions for use that are physically related such that they are considered a unit of manufacture, distribution, sale, or use.

The term "virtual packaging" means that the components of a kit are linked together by one or more physical or virtual instructions about the components that instruct the user how to obtain other components, e.g. containing one component Eppendorf tubes, bags, vials, or other containers and instructions directing the user to access a website, obtain record information or callback services, view visual information, or contact the manufacturer or seller on how to use the kit or guidance on its safety or technical information regarding one or more of the kit components. For example, the kit may contain one or more Eppendorf tubes containing one or more pairs of primers and instructions directing the user to access a website, obtain logged information or return services, view visual information, or contact the manufacturer. or the seller for guidance on how to use the kit and/or how to obtain technical information about other components of the kit.

The present invention also provides the use of the nucleic acid primer or primer combination as described in the first aspect in preparing a detection kit for identifying Yersinia pestis-specific phages in biological specimens.

3. Phage culture and identification methods

In a third aspect, the present invention relates to a method for identifying Yersinia pestis-specific phages in biological specimens, the method comprising the following steps:

a) grind tissue samples and perform nucleic acid extraction; and

b) Perform PCR detection on the nucleic acid sample extracted in step a) using the primer as described in the first aspect or the detection kit as described in the second aspect.

In one embodiment, the above method further includes the following steps:

c) Proliferate and culture the sample that is positive in the primer test in step b) in Yersinia pestis;

d) Separate the phage proliferation fluid; and

e) Phage lysis experiments in Y. pestis.

It should be understood that any technical features, embodiments, preferred embodiments and any combination thereof described in the first and second aspects above can be introduced into the embodiments of the third aspect, and the resulting embodiments of the third aspect are all are included within the scope of the present invention.

In step a), the processing of tissue samples and nucleic acid extraction are methods known in the art, such as the method described in Protocol: Purification of Total DNA from Animal Tissues (Spin-Column Protocol) disclosed in QIAGEN's commercial kit, See https://www.qiagen.com/us/resources/resourcedetail? id=68f29296-5a9f-40fa-8b3d-1c148d0b3030&lang=en.

In step b), PCR detection can be performed with reference to conventional methods in the art. In one embodiment, the PCR detection can use a 20 μL reaction system, which contains:

10μl commercial Premix,

8μl ultrapure water,

0.5μl each upstream and downstream primers (10μmol/L), and

1μl DNA to be tested.

In one embodiment, the PCR detection is performed using primers selected from:

F da:5’-ATgTCCggTgAATCTAATgCTg-3’

R da: 5’-CCAAAAgTCTTAgTACggCTTgC-3’

where F and R represent forward and reverse primers respectively; or

F xiao：5’-ggTgAAgAAgAAgACgAgAAT-3’

R xiao：5’-CTTAACggTCgCTAggTC-3’

where F and R represent forward and reverse primers respectively.

In one embodiment, the PCR detection is a nested PCR using two pairs of primers, including:

(1) Use the following first pair of primers to amplify the extracted tissue sample nucleic acid to determine the presence of the target fragment, where the amplified target fragment is 737bp,

F da:5’-ATgTCCggTgAATCTAATgCTg-3’

R da: 5’-CCAAAAgTCTTAgTACggCTTgC-3’

where F and R represent forward and reverse primers respectively; and

(2) Use the following second pair of primers to amplify the first round amplification product to determine the presence of the target fragment, where the amplified target fragment is 510 bp,

F xiao：5’-ggTgAAgAAgAAgACgAgAAT-3’

R xiao：5’-CTTAACggTCgCTAggTC-3’

where F and R represent forward and reverse primers respectively.

In one embodiment, steps c) to e) include the following steps:

(1) Add the tissue filtrate that was positive in the primer test in step b) to the freshly prepared Yersinia pestis bacterial solution,

(2) Shake the above mixture to mix it evenly,

(3) The resulting mixture is cultured statically at 28°C.

(4) Pass the cultured phage proliferation solution through a 0.45μm filter,

(5) Pass the obtained filtrate through a 0.22μm filter,

(6) Add the resulting filtrate to a freshly prepared Y. pestis-inoculated Buchner plate, and

(7) Observe the phage lysis to determine whether it is a Yersinia pestis-specific phage.

In one embodiment, the steps c) to e) include the following specific steps:

1. Bacterial liquid preparation: grind fresh Yersinia pestis from Brucella plate (90mm diameter) into 6ml Brucella broth and put it in a 50ml centrifuge tube;

2. Take 2 ml of the tissue filtrate that tested positive for the specific primer and add it to the above bacterial solution, and tighten the cap of the centrifuge tube;

3. Wrap the centrifuge tube containing Y. pestis and positive filtrate with gauze moistened with Lysol solution to prevent aerosol from escaping during shaking;

4. After shaking, place the centrifuge tube upright into the centrifuge tube rack and incubate at 28°C;

5. Centrifuge the phage proliferation solution that has been cultured at 28°C for one day at 5000 rpm for 5 minutes. After centrifugation, the supernatant is passed through a 0.45 μm filter;

6. Pass the filtrate from step 5 through a 0.22μm filter;

7. Use the filtrate from step 6 to conduct Yersinia pestis-specific phage lysis test;

8. Observe the phage lysis the next day. If the plague phage test is positive, the plague phage is successfully isolated. The result in step 6 is the Y. pestis-specific phage after proliferation and culture.

definition

Terms used herein have the following definitions. If no explicit definition is given herein, the terms used in the present invention have the meaning commonly understood in the art.

For the purpose of interpreting this specification, the following definitions will be used and, where appropriate, the terms "a", "an" or "the" used in the singular may also include plural referents and vice versa. It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not limiting.

As used herein, the term "and/or" means any one of the options or two or more of the options.

As used herein, the term "comprises" or "includes" means the inclusion of the stated element, integer or step, but does not exclude any other element, integer or step. When the term "comprises" or "includes" is used herein, it also encompasses a combination of the stated elements, integers, or steps unless otherwise specified. For example, when reference is made to a kit "comprising" a nucleic acid primer and instructions for use, this is also intended to encompass a kit consisting of the nucleic acid primers and instructions for use.

As used herein, "buffer" refers to a pH buffer. For example, the buffer may be selected from buffers formed from histidine, glutamate, phosphate, acetate, citrate and/or tris(tris).

The term "about" when used in conjunction with a numerical value is meant to encompass a range of numerical values having a lower limit that is 10% less than the specified numerical value and an upper limit that is 10% greater than the specified numerical value.

Primers can be prepared using any convenient synthetic method. Examples of such methods can be found in standard textbooks such as "Protocols for Oligonucleotides and Analogues; Synthesis and Properties", Methods in Molecular Biology Series; Volume 20; Ed. Sudhir Agrawal, Humana ISBN: 0-89603-247-7; 1993; 1st Edition ("Protocols for Oligonucleotides and Analogs: Synthesis and Characterization", Methods in Molecular Biology Series, Volume 20, edited by Sudhir Agrawal, Humana ISBN: 0-89603-247-7, 1993, Volume 1 Version). If desired, primers can also be labeled for easier detection.

Unless otherwise specified, the present invention can be implemented using conventional chemical techniques, molecular biology techniques, microbiological techniques, recombinant DNA techniques and immunological techniques, which are all within the capabilities of those of ordinary skill in the art. Such techniques have been described in the literature. See, for example, J. Sambrook, E.F. Fritsch, and T. Maniatis, 1989, "Molecular Cloning: A Laboratory Manual," 2nd ed., Books 1-3, Cold Spring Harbor Laboratory Press; Ausubel, F.M. et al. (1995 and periodic supplements; Current Protocols in Molecular Biology, ch. 9, 13, and 16, John Wiley & Sons, New York, N.Y.); B. Roe, J. Crabtree and A. Kahn, 1996, "DNA Isolation and Sequencing: Essential Techniques, John Wiley &Sons"; J.M. Polak and James O'D. McGee, 1990, "In Situ Hybridization: Principles and Practice"; Oxford University Press; M.J. Gait (editor), 1984, "Oligonucleotide Synthesis: A Practical Approach", IrL Press; D.M.J. Lilley and J.E. Dahlberg, 1992, "Methods of Enzymology: DNA Structure Part A: Synthesis and Physical Analysis of DNA Methods in Enzymology", Academic Press; and E.M. Shevach and W. Strober, 1992, regular supplement "Current Protocols" in Immunology, John Wiley & Sons", New York, NY. Each of these texts is incorporated herein by reference.

All patents, patent applications, publications, and other references cited or mentioned herein are incorporated by reference to the extent permitted by law. However, these references are discussed solely for the purpose of summarizing the claims made therein and are not an admission that any of these patents, patent applications, publications or other references, or any portion thereof, is relevant prior art to the present invention.

Description of the drawings

Figure 1 depicts the Yep-phi genome site map and the locations of some of the designed primers.

Figure 2 shows the results of PCR experiments using pure phage nucleic acid as the template for the test primers id29-1F/id29-1R, id29-2F/id29-2R, id43-1F/id43-1R, and id43-2F/id43-2R. As can be seen from Figure 2, the test primer id29-1F/id29-1R has low amplification efficiency, while the test primers id29-2F/id29-2R, id43-1F/id43-1R, id43-2F/id43-2R have high amplification efficiency but the product Shorter.

Figure 3 shows the results of test primers 325-402F/325-402R and 37714-37818F/37714-37818R in PCR experiments using pure phage nucleic acid as template. As can be seen from Figure 3, the test primer 325-402F/325-402R has non-specific amplification, the amplification efficiency of the test primer 37714-37818F/37714-37818R decreases as the annealing temperature increases, and the amplified fragment is short.

Figure 4 shows the results of test primers 1-331F/1-331R and 2-323F/2-323R in PCR experiments using pure phage nucleic acid as template. As can be seen from Figure 4, the test primers 1-331F/1-331R and 2-323F/2-323R have non-specific amplification, and the annealing temperature range of 2-323F/2-323R is narrow.

Figure 5 shows the results of test primers F da/R da in PCR experiments using pure phage nucleic acid as template and tissue samples as template. As can be seen from Figure 5, primers F da/R da have high amplification efficiency for tissue samples and pure phage at different annealing temperatures and different concentration gradients.

Figure 6 shows the amplification results of tissue samples of plague host using primers F da/R da. As can be seen from Figure 6, this primer performs well in terms of specificity and amplification efficiency.

Figure 7 shows the amplification results of tissue samples of plague host using primers F xiao/R xiao. As can be seen from Figure 7, this primer performs well in terms of specificity and amplification efficiency.

Figure 8 shows the amplification results of tissue samples by nested PCR, in which the first-round amplification primers are F da/R da, and the second-round amplification primers are F xiao/R xiao. The first round of DNA template is plague host animal tissue sample nucleic acid, and the second round of DNA template is the first round of amplification product. As shown in Figure 8, this primer combination performed excellently in terms of sensitivity, specificity, and amplification efficiency for phage detection and could meet the requirements.

Figure 9 shows the tissue sample amplification of plague host by primers F da/R da, F xiao/R xiao, 325-402F/325-402R, 37714-37818F/37714-37818R, and 1-331F/1-331R. As shown in Figure 9, the amplification efficiency of primers F da/R da and Fxiao/R xiao on tissue samples of plague hosts is significantly higher than that of primers 325-402F/325-402R, 37714-37818F/37714-37818R, and 1-331F. /1-331R.

Figure 10 shows an example of a field animal sample used for sampling, a Himalayan marmot that succumbed to infection with Y. pestis and was exposed to the wild environment.

Figure 11 shows the results of the lysis test of the Yersinia pestis-specific phage. The Yersinia pestis-specific phage identified and isolated using the method of the present invention has a titer of 3x10 ⁹ PFU/ml.

Detailed ways

The present invention will be further illustrated by examples below, but the following examples should not be construed as limiting the invention.

Example 1: Design and verification of specific primers

1. Primer design

The primers were designed to target the unique sequences of Yersinia pestis-specific phages Yep-phi and YepMm. A large number of primers were designed for their unique sequences, and preliminary verification was conducted on phage nucleic acids, sample nucleic acids from which phages were isolated, and sample nucleic acids from which no phages were isolated. After multiple rounds of screening, some promising candidates were obtained. Table 1 below lists some exemplary candidate primers for further screening.

Table 1. Phage detection primer information for PCR validation

2. Primer screening using pure phage nucleic acid as template

This experiment uses pure phage nucleic acid as a template to test candidate primers and screen out ideal amplification primers.

1. Exclude primers with low amplification efficiency, non-specific amplification, or narrow annealing temperature (Experiment 1)

Method: Use 20 μl amplification system, 10 μl commercial Premix, 8 μl ultrapure water, 0.5 μl each of upstream and downstream primers (concentrations are 10 μmol/L, see table 1 for sequences), and 1 μl of phage nucleic acid as DNA template. The amplification program was: pre-denaturation at 95°C for 5 min; 25 cycles of 95°C for 15 s, annealing for 15 s, 72°C for 30 s; and extension at 72°C for 5 min. The gradient of annealing temperature is 53.3℃, 54.2℃, 55.3℃, 56.4℃, 57.6℃, 58.7℃, 59.8℃, 60.7℃.

Results: The target nucleic acid sequence of the test primer id29-1F/id29-1R is the major head protein and tail protein of the phage, located in the reference sequence Yep-phi (Genbank Accession: HQ333270) 21,117...21,604, and the target fragment length is 488 bp. Compared with other test primers, the overall amplification efficiency of id29-1F/id29-1R is low, and the amplification efficiency drops significantly at 58.7°C-60.7°C (Figure 2, id29-1). The target nucleic acid sequence of the test primer id29-2F/id29-2R is the phage major head protein and tail protein, located in the reference sequence Yep-phi (HQ333270) 21,187...21,496, and the target fragment length is 310bp (Figure 2, id29-2); test The target nucleic acid sequence of primer id43-1F/id43-1R is phage hypothetical protein, located in the reference sequence Yep-phi (HQ333270) 37,876...38,163, and the target fragment length is 288bp (Figure 2, id43-1); the test primer id43-2F/ The target nucleic acid sequence of id43-2R is a phage hypothetical protein, located at reference sequence Yep-phi (HQ333270) 37,841...38,196, and the target fragment length is 356bp (Figure 2, id43-2). The test primers id29-2F/id29-2R, id43-1F/id43-1R, and id43-2F/id43-2R have high amplification efficiency and no non-specific bands, but the amplification product length is low and is not suitable for nested PCR. amplification.

The target nucleic acid sequence of the test primer 325-402F/325-402R is located in the non-functional annotated region of the phage reference sequence Yep-phi (HQ333270) 225...786. The target fragment length is 562bp, and there is non-specific amplification around 1000bp (Figure 3, 325 -402). The target nucleic acid sequence of the test primer 37714-37818F/37714-37818R is the hypothetical protein of the bacteriophage, located in the reference sequence Yep-phi (HQ333270) 37,616...37,885. The target fragment length is 270bp. The annealing temperature is 59.8°C and the amplification efficiency decreases at 60.7°C ( Figure 3, 37714-37818). The target nucleic acid sequence of the test primer 1-331F/1-331R is located in the repeat region of 39...447 of the phage reference sequence Yep-phi (HQ333270). The target fragment length is 409 bp. There is non-specific amplification and it is obvious as the annealing temperature increases (Figure 4, 1-331). The target nucleic acid sequence of the test primer 2-323F/2-323R is located in the repeatregion 68...468 of the phage reference sequence Yep-phi (HQ333270). The target fragment length is 401bp. The overall annealing temperature range is narrow. As the annealing temperature increases, the amplification efficiency decreases significantly. , there is no amplified band at 59.8℃ and 60.7℃ (Figure 4, 2-323).

Therefore, the above test primers cannot be used as ideal primers for phage detection due to low amplification efficiency, non-specific amplification, narrow annealing temperature range and other factors.

2. Primers F da/R da have the advantages of high amplification efficiency, no non-specific amplification, and wide annealing temperature range (Experiment 2)

Method: Use 20 μl amplification system, 10 μl commercial Premix, 8 μl ultrapure water, 0.5 μl each of upstream and downstream primers Fda/R da (concentrations are 10 μmol/L, see table 1 for sequences), DNA template is 1 μl phage nucleic acid, phage The nucleic acid concentrations are 1ng/μl, 0.1ng/μl, 0.01ng/μl, 0.001ng/μl, and 0.0001ng/μl. The amplification program was: pre-denaturation at 95°C for 5 min; 25 cycles of 95°C for 15 s, annealing for 15 s, 72°C for 30 s; and extension at 72°C for 5 min. The gradient of annealing temperature is 52°C, 54°C, 56°C, 58°C, and 60°C.

Results: The target nucleic acid sequence of primer F da/R da is located in the head assembly protein of 19,197...19,933 of the phage reference sequence Yep-phi (HQ333270), and the target fragment length is 737 bp. For pure phage nucleic acid, primers Fda/R da have high amplification efficiency in the annealing temperature range of 52-60°C without non-specific amplification. The amplification efficiency of phage DNA template at the same concentration is consistent at different temperatures, and the detection limit of pure phage nucleic acid by primers F da/R da is ≤1x10 ^-4 ng/μl (Figure 5, rows 3 and 4).

Primers F da/R da have a wide annealing temperature range, making them suitable for both ordinary PCR and nested PCR. When the primers are used for ordinary PCR detection, a higher annealing temperature can be used to improve the specificity of the detection (as shown in Experiment 4 below); when the primers are used for the first round of nested PCR amplification, a lower annealing temperature can be used to improve the specificity of the detection. The sensitivity of the detection (as shown in Experiment 6 below). As the first-round primer of nested PCR, F da/Rda has a moderate product length of 737 bp. Designing a nested second-round primer within the first-round product can prevent the second-round product from being too short. The amplified fragment only has 100% consistency for phages Yep-phi and YepMm, and the consistency of other phages with this fragment is 98.91% or less, thus ensuring the specificity of the detection results for Yersinia pestis phage.

3. Use tissue sample nucleic acid as template to verify primer F da/R da

1. The lower detection limit of primers F da/R da for tissue samples containing phages is 1x10 ^-2 ng/μl (Experiment 3)

Method: Use 20 μl amplification system, 10 μl commercial Premix, 8 μl ultrapure water, 0.5 μl each of upstream and downstream primers Fda/R da (both concentrations are 10 μmol/L, the sequence is shown in Table 1), and the DNA template is a tissue sample containing phage. Nucleic acid 1μl, sample nucleic acid concentration is 1ng/μl, 0.1ng/μl, 0.01ng/μl, 0.001ng/μl, 0.0001ng/μl. The amplification program was: pre-denaturation at 95°C for 5 min; 25 cycles of 95°C for 15 s, annealing for 15 s, 72°C for 30 s; and extension at 72°C for 5 min. The gradient of annealing temperature is 52°C, 54°C, 56°C, 58°C, and 60°C.

Results: For tissue sample nucleic acids containing phages, the amplification efficiency of primers F da/R da was high in the annealing temperature range of 52-60°C, without non-specific amplification. The amplification efficiency of tissue sample DNA templates of the same concentration at different temperatures is basically the same. The lower limit of nucleic acid detection for phage-positive tissue samples with primers F da/R da is 1x10 ^-2 ng/μl (Figure 5, rows 1 and 2).

2. Conduct large-scale verification using tissue samples from plague host animals

The test objects are tissue samples that may contain plague-specific phages, such as heart, liver, spleen, lung, kidney, bone and other samples of plague host animals.

1) Ordinary PCR: primers F da/R da amplify tissue samples (Experiment 4)

Method: Use 20 μl amplification system, 10 μl commercial Premix, 8 μl ultrapure water, 0.5 μl each of upstream and downstream primers Fda/R da (concentrations are 10 μmol/L, see Table 1 for sequences), and the DNA template is plague host animal tissue sample. Nucleic acid 1μl. The amplification program was: pre-denaturation at 95°C for 5 minutes; 25 cycles of 95°C for 15 seconds, annealing at 59.5°C for 15 seconds, and 72°C for 30 seconds; extension at 72°C for 5 minutes.

Results: Tissue samples were detected using primers F da/R da alone. The amplification band of phage-positive samples was single without non-specific amplification. The intensity of the band varied with the phage content. Samples with lower loading capacity can be detected (Figure 6, row 1, 54x).

2) Ordinary PCR: primers F xiao/R xiao amplify tissue samples (Experiment 5)

Method: Primers F xiao/R xiao are located in the head assembly protein of phage reference sequence Yep-phi (HQ333270) 19,380...19,889, and the target fragment length is 510bp. Use a 20 μl amplification system, 10 μl commercial Premix, 8 μl ultrapure water, 0.5 μl each of the upstream and downstream primers F xiao/R xiao (both concentrations are 10 μmol/L, the sequence is shown in Table 1), and the DNA template is plague host animal tissue sample nucleic acid. 1μl. The amplification program was: pre-denaturation at 95°C for 5 minutes; 25 cycles of 95°C for 15 seconds, annealing at 59.5°C for 15 seconds, and 72°C for 30 seconds; extension at 72°C for 5 minutes.

Results: The tissue samples were detected using primers F xiao/R xiao alone. It was seen that the amplification band of the phage-positive samples was single, and there was no non-specific amplification of the phage-negative samples (Figure 7).

3) Nested PCR: Use primers F da/R da and F xiao/R xiao to perform nested amplification of tissue samples (Experiment 6)

The first round amplification primer of nested PCR is F da/R da, and the first round amplification product is used as the template for the second round amplification. The second round amplification primer F xiao/R xiao is located in the first round amplification product. inside, further improving the sensitivity of detection.

Method: Use 20 μl amplification system, 10 μl commercial Premix, 8 μl ultrapure water, 0.5 μl each of the upstream and downstream primers (concentrations are 10 μmol/L, see Table 1 for sequences), and the DNA template is 1 μl nucleic acid from plague host animal tissue samples. The amplification program was: pre-denaturation at 95°C for 5 min; 25 cycles of 95°C for 15 s, annealing for 15 s, 72°C for 30 s; and extension at 72°C for 5 min. The first round of DNA template is plague host animal tissue sample nucleic acid, and the second round of DNA template is the first round of amplification product. The first round of annealing temperature is 54.4°C, and the second round of annealing temperature is 59.5°C.

Results: Nested PCR was used to detect phages in tissue samples. There was no non-specific amplification in both rounds of amplification, and the positive sample amplified a single band. The positive sample "71 bones" was detected in the first round (Figure 8, first row), and the positive samples "34 bones", "34 mandibles", and "71 bones" were detected in the second round (Figure 8, second row) , nested PCR can further improve the sensitivity of phage detection, and is suitable for the detection of tissue samples with low phage content, further reducing the detection limit.

4. Use plague host tissue sample nucleic acid as template to verify other primers

In experiments using tissue sample nucleic acids as templates to verify other primers, primers F da/R da and F xiao/R xiao were used for comparison. The inventor found that the detection effects of other primers were not as good as those of primers F da/R da and F xiao/ R Xiao ideal. Several other primer pairs are given here as examples.

Ordinary PCR: primers F da/R da, F xiao/R xiao, test primers 325-402F/325-402R, 37714-37818F/37714-37818R, 1-331F/1-331R for tissue sample amplification (Experiment 7)

Method: Use 20 μl amplification system, 10 μl commercial Premix, 8 μl ultrapure water, 0.5 μl each of upstream and downstream primers (concentrations are 10 μmol/L, see table 1 for sequences), and the DNA template is 1 μl nucleic acid from plague host animal tissue samples. The amplification program was: pre-denaturation at 95°C for 5 min; 25 cycles of 95°C for 15 s, annealing for 15 s, 72°C for 30 s; and extension at 72°C for 5 min. Annealing temperature: The annealing temperatures of primers F da/R da and F xiao/R xiao are the same as in Experiment 6, which are 54.4°C and 59.5°C respectively. According to experiment 1 (Figure 3, Figure 4), the annealing temperature of the test primers was 53.3°C.

Results: The amplification efficiency of tissue samples using primers F da/R da or F xiao/R xiao is the highest. It can be seen that the amplification band of phage-positive samples is single, and the amplification results of phage-negative samples are negative and there is no non-specific amplification.

Compared with primers F da/R da or F xiao/R xiao, the amplification efficiency of test primers 325-402F/325-402R is relatively low; test primers 37714-37818F/37714-37818R, 1-331F/1-331R The amplification efficiency dropped significantly, and the test primer 1-331F/1-331R caused non-specific amplification of some samples.

The results show that, as shown in Figure 9, primers F da/Rda or F xiao/R xiao have obvious advantages over other primers in terms of amplification efficiency and specificity.

Example 2: Phage isolation and culture

1. Preparation of phage filtrate

Sample: Phage PCR-positive tissue immersion fluid (immersed in physiological saline, frozen or temporarily stored at 4°C)

1. Thaw the tissue immersion solution at 4℃.

2. Take 2 ml of tissue immersion fluid, pass it through a 0.45 μm filter first, and put the filtrate into a sterile tube.

3. Pass the obtained filtrate through a 0.22 μm filter, and put the filtrate into a sterile cryovial to obtain the phage filtrate.

4. Store the phage filtrate at 4°C until use.

2. Phage Proliferation

Sample: Phage Filtrate

1. Preparation of bacterial solution: Grind fresh Yersinia pestis from Brucella plate (diameter 90mm) into 6ml Brucella broth and put it in a 50ml centrifuge tube;

2. Add all the phage filtrate to the above bacterial solution and tighten the centrifuge tube lid;

3. Wrap the centrifuge tube containing Yersinia pestis and the positive filtrate with gauze moistened with Lysol solution to prevent aerosol from escaping during shaking;

4. After shaking, place the centrifuge tube upright into the centrifuge tube rack and incubate at 28°C;

5. Centrifuge the phage proliferation solution that has been cultured at 28°C for one day at 5000 rpm for 5 minutes. After centrifugation, the supernatant is passed through a 0.45 μm filter;

6. Pass the filtrate from step 5 through a 0.22μm filter, and put the filtrate into a sterile tube. The result is the multiplied phage filtrate.

7. The proliferated phage filtrate is used for subsequent phage lysis experiments, phage plaque experiments, etc., for further qualitative and quantitative detection of phages.

3. Phage lysis test

Perform the plague phage lysis test according to B.4.1 of "Plague Diagnostic Standards/WS 279-2008". After testing, all positive samples detected by the PCR reaction with the above primers F da/R da and F xiao/R xiao were positive in the plague phage lysis test after phage culture and proliferation, proving that the plague phage was successfully isolated, and the primers The detection results of F da/R da and Fxiao/R xiao for target phages are very reliable.

For example, as shown in the lysis test in Figure 11, the Yersinia pestis-specific phage identified and isolated using the method of the present invention has a titer of 3x10 ⁹ PFU/ml.

sequence of the invention

SEQ ID No.1: HQ333270.1 Yersinia phage Yep-phi, complete genome

CTAATAGTACATACAAAGTGTTTCCCCCCTATGGCCTCCTTATGCTCACCCCAAAGAGAGCACCCTATGGTGACTTTA

GGTTTACTTTAGGTGTACTTGAGGTGTACTTTAGGTGTACTTAATGCCTAACCTTATGTGTGACATTGGGTTTACTACTA

GATGTAGTGCTTAGGGGGGACTTTAGGGGACTATATGTAGGCCTCTTTCTGTCTCTGACTGTAAGGGACCCTCTGAGTA

TCTCCTTATGTCACCACCTTAAGTCCTCTCCTTATGTCACCACCTTATGTCACCACCTTAAGTCCTCTCCTTATGTCACC

ACCTTATGTCACCACCTTAAGTCCTCTCCTTATGTCACCACCTTATGTCACCACCTTAAGTCCTCTCCTTATGTCACCAC

CTTAAGTCAGCCTCTATCCTTATGTTAACCTTCAGTCGGTGCCTTCAGTCTATAGACCTTATGTTACCACTCTTAGAGAG

ACTACTTAGAGGCTACTTAGAGACTACTTATAGGCTACTTAGAGACTACTTATAGGCTACCTAGAGACTACTTATAGG

CTACCTAGAGAGACCCTTACAGTTAATACGACTCACTATTGGGAAGGTGTCTATTACTATGCCCTTCCTTATAGGTATA

ACTTAGAGTGCTAACTTAAAGAACATTCAAAGAACACTCAAAGAACACTTAAAGATGACTTTTTGTTACTTTAAGTAT

TGACTCTAAGATTAGATACTGTATTATTCATCTCACACCAACACAGTGAGTCGCCCTCTGAAACAACATGAGGTGTCA

AGGTGACAATCACTAGGTTGACAAGCAGTAACACTAAGACGTACAATGATTCACACAGATTCACATAGATTCACCGCT

CTTTAAAAACTTGGATAGTAACACTCTTGAGAATGACCAATAGCGATTAACTCAAGGTGACTCGAAGAGAGAAGCCTT

TATGATAATCACTAACTTACTAACTAATGAGGTCATCACACTATGAACGAATACTCTGGCTGGTTCTTCACATATAACT

CATTCTCTAGGTACTTTGGGACACATCATTTGACAGGTCTAACGCCACTAGGTGTAGTGACTGCTGCTCTCTTAGTCAT

CGCAATGTATAAGGCAATCAAGTATTGGCTTTAACTACTATCGCGGTAAGCAGCTCGCTAAGTCTCTAATTGTTCCTGA

AGGGCCTCATAAAGGGTCTGAGGATACACAAGGCTAACCTCTAGCACTTGAAGCATGGCCTGCTCTCTACAACTAACC

TTCTACTATCTTAAATAGGACACACTCTAATGAACTACACCGACATTCAGGCACGTCTAGCAATCATCAAGTCTCTCCC

TATTAGAGAACTGGACAAGCGCCAGCCTCTTCTGGTTGAACTAGCTGCTGACATTGTGAACGGTGAGACTAATAACGG

TAACGATACAGATGGGTCTAAGGGTCTTGAGTATCAGGACTGGTGGCATATCTTAGGGTCACTCATGGGTGATGCAGG

GTTCAACCTGTTAGGTAATGGTCACTTCAGTGCAGCTTACTCACACAAATGTTACCCGGTCGAGTCATTAAGGTTGGC

TTTAAGAAAGAAGACTCAGGAGCTGCATACACTGCGTTCTGTAGGATGCACCAAGGTCGTGCTGGTATCCCTAACATC

TATCACGTAGCACGTCACGCAGGCTGCTACACAGTTGTACTTGACCATTTGAGTGACTGTAAGCGAGACACTAATGAG

ACTCACGCTAACTATGCGTATGCTGCAAGGGCACTCATAGAATGTAACGATATCAATGTCGAAGCTTGTGCACATTTTT

GTTAGCACTGAGTTCATCGAGACGTGCAAGATGATTCGTGAGTTCTTCTACGGGATTGCGTCTTTCGATATGCACAGTG

GGAACATTATGTTCGACAAGAACGATGTGGCTTACATAACTGACCCTGTTAGCTTCTCTCATGACCGTGAACGTGAGG

ATGGCTTCCCGTTGGACCCTGATGCACTGATTGCTGAGGTCGAGGCGATAGCTAACGAGCGTATCATCGAACGTTGTC

GGAACCGTAAGGCAAAGCGTGACCCTAATGGTACATTCCGTGCTGAACATAAGGCTGCAATGAAACGCCGTAAGCGT

AACCGTAAGTTATCTGAATGTAGAGCTGAACGGGTAGCTAAGTGTGAAACTAAGCAGGACCACTTACACTTAATGTCC

CTCCAAAGGGAACACAAAGTAATCGAACGAAACGAACGTCGTGCTGAGATGCTCATGGGGTCTGTATGGCATGACTT

CAGGTTACTCAATGGGAACTCAACGATACACAAGGTTAACCAAGTGAACGACTTTAAGTGGCAAGTAGCTGACGGTC

TGGCTATTCAGGCAGGATGTTCACTAATGATTGATAAAATACTTGACGCACAACTGATGGGATGATAAGGTGTATCTT

CAAGGTCGGTACTAACTGCTGGCCTTTATAGATAAACTTTCTTAACAACGTGAGGCAACAATGACTAACGTAATCAAC

GCACCAAAGAACGACTTCTCTGATATCGCTAATGCAATCCAGCCGTACAACATCCTGGCTGACCATTACGGTGCACAG

CTTGCAGCTACACAGCTTGAGTTGGAACATGAAGCGCACACTGAGGGCGAGAAGCGTTTCCTTAAGGCAATGGAGCG

TCAAATCAAAGCAGGTGAGTTCGGTGATAACACGGTAGCTAAACCGCTCCTGTCATCCCTTGCACCAAAGTTTATCGA

AGCGTGGAACACTTGGTTCATTGATGTAGAAGCAAAGCGTGGTAAGCGTCCAGTGGCCTACAATCTGGTACAGAAGG

TTGCACCTGAAGCTGCTGCTTTCATCACTCTGAAAGTAACACTGGCTTGCTTGACGAAAGAAGAGTTCACCAACCTGC

AGTCGGTAGCCACTAAGATTGGCCGCAGCATTGAGGACGAGTTACGCTTTGGGCGCATCCGTGACGAAGAAGCGAAG

CACTTTAAGAACCACGTACAGGAAGCACTGAACAAGCGCGTTGGTATCGTCTACAAGAAAGCATTCATGCAAGCTGT

AGAGGGCAAGATGCTCGATGCCGGTCAACTGCAAACCAAATGGACTACTTGGACACCTGAAGAAGTAATTCACGTTG

GTGTACGTATGTTAGAGTTACTCATTGGGTCTACTGGTCTCGTTGAGTTGCACCGTCCGTTTGCAGGCAACATTGAGAA

AGACGGAGAGTACATCCAGTTGACCGAACAATATGTTGACCTACTGTCTAAACGTGCTGGTGCACTGGCTGCAATTGC

TCCAATGTATCAACCTTGTGTAGTCCCTCCTAAGCCTTGGACTTCTCCAGTAGGTGGTGGCTATTGGGCTGCTGGTCGT

AAGCCTCTCTCTATGGTCCGCACTGGTTCCAAGAAGGGCTTAGAGCGTTACAACGATGTGTATATGCCTGAAGTCTAC

AAGGCAGTGAACATCGCACAGAATACTCCTTGGAAGATTAACAAGAAGGTTCTGGCAGTGGTCAACGAGATTGTGAA

CTGGAAGCATTGCCCGGTTGCTGACGTACCTGCTATGGAACGTGGTGAACTACCAGTTAAACCTGTAGACATCGATAC

GAACGAAGCGTCTCTCAAAGCGTGGAAGAAAGCAGCATCTGCAATCTATCGTAAAGAGAAGGCTCGTGTGTCTCGTC

GTATGAGCATGGAGTTCATGTTAGGTCAGGCCAATAAGTTCGCACAGTTTAAAGCTATCTGGTTCCCTATGAACATGG

ACTGGCGTGGTCGTGTATACGCAGTACCAATGTTTAACCCACAAGGTAACGACATGACTAAAGGTCTGCTGACATTAG

CTAAAGGTAAACCAATTGGTGTTGATGGGTTCTACTGGTTGAAGATTCATGGTGCTAACACTGCTGGTGTTGACAAGG

TGGACTTCGCAGAACGCATCAAGTTCATTGAGGACAACCATGAGAACATCATGAGCGTTGCAGCTGACCCTATTGCTA

ACACTTGGTGGACTGAACAAGACTCACCGTTCTGCTTCCTCGCGTTCTGCTTTGAGTATGCAGGTGTGCAACATCACGG

GATGAACTACAACTGCTCTCTGCCTCTGGCGTTCGATGGTAGCTGCTCTGGTATTCAACACTTCTCTGCAATGCTGCGT

GACGAAGTGGGTGGACGTGCTGTTAACCTACTGCCAAGCAAAGAAGTACAAGACATCTATCGTATCGTAGCTGAGCG

TGTGAACGAGATTCTTAATCAAGATGTAATCAACGGTACTGACAACGAAGTTGAAACACTGACCAATAAGGACACTG

GAGAAATCACTGAGAAGCTCAAGCTGGGGACTAAAGAACTGGCTGGTCAATGGTTGGCTTATGGTGTAACACGTAAG

GTAACTAAGCGTTCCGTAATGACCTTGGCATATGGTTCCAAAGAGTATGGCTTCCGTGACCAAGTGTTAGAGGATACC

ATTCAGCCAGCTATCGACGATGGCAAGGGCTTGATGTTCACTCAACCTAACCAAGCCAGCTGGCTACATGGCTAAACTG

ATTTGGAATGCAGTAACAGTCACAGTGGTTGCAGCAGTTGAGGCAATGAACTGGTTGAAGTCCGCAGCTAAACTTCTG

GCTGCTGAAGTTAAAGACAAGAAGACTAAAGAGGTTCTTCGTAACCGTTGCGCTGTATACTGGGTAACTCCAGATGGC

TTCCCTGTATGGCAGGAGTATCGCAAGCCAGTACAGACCCGCTTGAACTTAATGTTCTTAGGTCAGATTCGTCTACAAC

CTACTGTGAACACCAATAAGGATAGCGGAATTGATGCGCGTAAGCAGGAGTCAGGCATCGCACCTAACTTTGTCCACT

CAATGGATGGAAGTCACCTTCGAATGACTGTAGTTCGCTCCTATGAGGTCTACGGTGTGGAATCCTTCGCACTGATTCA

TGACTCATTTGGTACTATCCCGGCTGATGCTGGTAACCTGTTCAAGGCTGTACGTGAAACAATGGTGAACACCTACGA

AGAAAACGATGTGCTTGCAGACTTCTATGACCAGTTTGCTGATCAATTACATGAATCACAGCTTGATAAGATGCCTGA

GATGCCAGCTAAAGGGTCTCTAGATATCCAAGAAATCCTCAAGTCAGACTTTGCGTTTGCCTAACGGAATTTAATACG

ACTCACTATTGGGAGACCCAAGGGTTTCCCTACTTTAAGATACACTTTAAGATATACTCTAAGATGCACTTTAAGTTTA

CTTTAAGAGGAAACAACATCATGCGTACCAACTTTGAACCAATCACTAAGCGTAACAACGTAATCAACGAACATGGT

ACTGAATGGCAGGAACGTAAAGACCACATGAAGAAACGCCACAAGACACAGCGTGGTAACTCTGAGAAACGAAACT

GGAAGGAGGCACTCTAATGAGTAGCTTATATAGTGGGAACCTAAACGCCTTCAAGGCTGCTACTGACAGAATCTCTAA

GATGGACTTAGTGGTCGCTTGTGAGGAGTGGTATGAGTCACATGTAAGTCTGAACTGTATGTGGCTACGCATAGAGGA

CAAGGAAGGTAAGCGGATTAACTCCATCACTTGTAGTCATCACGACGAAGACGTTCTATACAGTATGGGAACCGAGT

GGTTGAACCGAATGTATGACCAGTTGAAAGACTGGAAGTAATACGACTCACTATTGCGAAAGAGTTAAACTCAAGGT

CATCTAAAAGGTGGCCTTTGTGATTAACTTTTTGATTAACTTTTGATTAAACCTTAACCAACACACACACATATACATC

TATAGGAGTTCCACAATGGAAATCACATTCAAGACTTCACCACATAAAGCTGTTTCATTCGTTGAGTCTTCCGTTAAGA

AAGCTCTCGACAAAGCTGGCTACCTTATCTGCGATGTAAAGTATGATGGTATTCGTGGTAACATTGTAGTTGACAATA

CGGCTGACAGCATGTGGCTCTCCCGTGTCTCTAAGACCATCCCTGCCCTTGAGCACCTCAATGGGTTCGACTCTCGCTG

GCAGCGTCTTCTCAAAGATGACCGCTGTATCTTCCCTGATGGTTTCATGCTTGATGGTGAACTGATGGTCAAAGACGTA

GACTTCAACACAGGGTCTGGCCTTCTGCGTACCAAATGGAGTGACCCTAAGAATTTCAGTACCAACTGTAACCCTTTG

GCCTCTGACTTCACTAAGAAGTCTGCCAAGGTTCCCTACATGTTGTCCTCTGATAAGCTTAAGGTTGTTCTTTATGGTG

TCATGCCGTTTGACTCTATGGTTTCCGGTGAGACCTACGAAGTGATGAACCTACTGATGCGTGAGCACGTTAAGGCTA

TGCTTCCGTTACTGAACGAGTACTTCCCTGAAATCAAATGGGACATCTCTGAGAGCTATGAAGTCTACGACTATGTGG

AACTGACAGCACTCTATGAGAAAGCTCGAGCAGATGGTCAGGAAGGTCTAGTAGTGAAAGACCCTCTGGCATTCTATC

AGCGTGGTAAGAAGTCTGGCTTCTGGAAGCTAAAGCCTGAGTGTGAAGCAGATGGTATCATTCAGTCTGTCAATTGGG

GAACACCGGGTCTATCTAATGAAGGCTTGGTGATTGGCTTTAATGTTCTCTTAGAGACTGGTCGGCATGTTGCAGCTAA

CAATATCTCACAGACTCTTATGGAAGAGCTTACAGCTAACGTTAAGGAACACGGTGAGGACTACTACAATGGATGGG

CCTGTCAGGTAGCCTACATGGAGGAAACAAGTGACGGGTCATTACGGCACCCCTCATTCGTTATGTTCCGTGGTGTTG

AGTCAGATCCAATGGTCAAGATGTAAATCACTCAATGGTCAGTCCTTAACAGGGGCTGGCCTTTTTGCGTTTCTAATTA

GACTCACTATGGAGGAACAACCACATGCTATTCCTAATTGTTGCTATCACTGCTGTCACACTTGCCTTAGTTATCGTTG

ATGACAACTGCTGGCCTGATTGCTAATACGACTCACATATGGAGGACACACATATGCTACGATTACACTTCAATAAAT

CTAATGGTATATACTCGGTTCGTAACTCAGGCCGCTCGCTGGTCAAGGCTACCGAGAAACATGCAGTCATTCCAAAAG

TGCTTGGTGCTACCATTGAGTTAGCACCCCGTGTTCACGCTGTGATTACTACTGGCTTATTTAAAGAAGCGACAACTGG

CTCCCGGCCTTTCATTCCGGTTCTCGTCACCCGTTTCCCTAAAGTCCGTCTGGTCATAGAGTGTATCAAGGAGGTGTTC

TAATGTATGGTTGCACTGCATGTATAGAGAATGATGACCGCTACCCACATACCTGTGAACCTGTAGCTAAACCACAAG

ATATAGATGGTGTTCGCACTCCGAGCCACTATATGTTGTTTGATGACATTGAGGCTATCGAAGTGATTGCCCGGTCGAT

GACCCGTGAACAGTTCAAAGGGTACTGCTTAGGGAACATCCTTAAGTATCGACTACGTGCTGGTAAGAAATCCGAGCT

GGCATTCTTAGAGAAGGACATGGCGAAAGCTGGGTTCTATAGTGAACTGTATGAGAGACATAAAGGTAAATGTTATG

CTTAAACCTAGTGAATGGTGTGAGAGAAAATTCAACGAGACTGGCAATGTCGACTATCTCGAAATGTACAACCTGTGG

AAAGGGAGAGGACAATGAGCATTTTAAACAAAGGGTCACTGGTCAACGGCGACAAGAAGTTCTTCGCTACTGTAGAA

GGCCAAGGCCAATCATTCGAAGTACCTGTGTTCGCTACATCTCTTGAAGAGGCCAACGAGTTAGCTGAGTGGCAATAT

GTCCCAGCTGGCTTCGCAGTGACACGCATTCGTCCAGACCGAAAGGTTTAATAGGACTCACTAATGGGAGACACATAG

TTTCCCACTTTTAACTTTCAGAAACTTAAGGAGAAACATCATGGCTTTCACTAAACGTAAAGTATTCACTTCTGCTCGT

GGTATTGCTGAACCGTACTGCTACCTGGCTAAGCCTGACTTTGGTAGTGGTGACTTCAAGAACGAACGTGGTGTCTAT

AAAGTATCACTAACTGTATCCAACGATGACCCTCGCTGTCAGAAAATGATCGATGAGATTGTCGAAGCCCATGAGACG

GATTATGCTGCACGTATGGAAGAGTACGAAGCTAACCCGCCGAAAGTTGTTAGGGGTAAGAAACCGCTGAAGCCATA

TGTTGGTGACATGCCTTTCATGGATAACGAAGATGGAACCACAACGTTCACCTTCAAGTGCTATGGTTCATACACTGA

CAGGAAGACTGGTGAGAATAGACCAATCGATTTAGCTATCGTGGACTCTAAGGGCAAGCGTATTCGTGGTGAGCGTCC

AGCTATCTCTGGTGGCTCCGAGCTGAAGATTAAATACACTCTGTTCCCTTATGGTTGGTCTGCTGTGGCAGGTGCTTCC

GTTAAGCTACAACTGGATTCCGTGATGCTGATTAAGCTGGTTGAGTTCGGTGGTAGCGGTGAGGATGACTGGGCTGAT

GAAATCGAAGAAGATGGTTATGTTGCTGTTGAAGCACAGACCCGTAAGCCTCAGAACGACTCTGGCTGGGATGAAGA

ACCAGAAGAGGACGATGAAGATGATGATGAGTCCGGCGACTTTTAATATATGATAGGGAGGTCACATGGCAGGTTAC

GGTGCTAAAGGAATCAAGAAGGTAGGTATCTACCGAAGTGGACTCGAAGAGAAGAACATGAAACTCTTAGAGTCCAA

GGGTGTTAAAGCTGAGTACGAAATGTGGAGAGTTCCTTATGTGGTTCCTGCAAGTAATCATCATTACACACCAGACAT

TTTATTACCTAACGGTATCTTTGTGGAGACAAAAGGCTTATGGGAAAGTGATGACCGCAAGAAACACTTGCTGATTCG

GGAACAGACCCCGACCTGGATATACGTCTGGTCTTCTCAAGCAGTCGCACCAAGATTTACAAGGGGTCTCCTACCAG

TTACGCTGAGTTCTGTGAGAAGCACGGCATCTTATTTGCTGACAAGCTGATTCCAGTATCCTGGTTGAAGGAAGCCTCC

CGGTCTGTACCGTTTGATAAACTGAAGACTAAGAAGGAGAAGAAGTAATGCCAAAGGTACAATTTAAACCACGCAGT

TCTACAGAGGCAATCTCGTACACTGTAGCGCAACAAAGCCAACAATGGACGTAGGTGTTCGTGAAATCAGTCAGTGG

CATAAAGAACAAGGTTGGTTAGCAATCGGCTATCATTTTGTGATTCGCCGTGATGGAACCATTGAGGAGGGACGTCCA

GTCGAAGTCATTGGGTCTCACGTTAAGAGCTGGAACAGTAAGTCAGTTGGTGTGTGTCTCGTTGGTGGTATTGACGAT

AAGGGCCACTTTGAGGCAAACTTTACGCCTGCTCAGATGGTCTCTCTTAAAGAGAAACTTGCAGACCTACTTGATATG

TATCCAGATGCTGAGGTGAAAGCTCACCATGACGTCGCACCTAAAGCGTGTCCGTCCTTCAACTTGAGCCGCTGGCTG

AAGACTGGAGAAATGGTCACAAGCGATTGGGGCTAATTAATAGAACTCACTATGGAAGAGCCGAAAGGTTTCTCCCT

TTGTTCGCTAAAAATGATTAAGGAGTAACCACAATGTATAACCTTAAAGAGCATGTGTTCGTCATTTTAATCTGTGTAC

TTATCATTGGTGGAGCCTGCCTGCGTCCGTTCCTATAGGGGGTATCTAATGGATATGGAAGAACAGCAAGATAGCGTG

TTCCTGTATCACCTGCCGTGTGAAAACTGTGGGTCGTCAGATGGTGACTCAATGTACTCCGATGGACACAGGCACTGT

TTCGTGTGTAACAACTATGTGCCTCCCACGGATGAGACTCGTGAGAAACTCTCCAAGCGGAAACGCTCATATAACACT

GGAGGTGACAAAATGTCTAACCTGTTAAACTTTGGGGATTCAGATGGTCGCTATACGAACCTCAAAGCTCGTGGCCTT

ATGGAAGCTATCTGCCGTAAGTATGGCTATTGGGTAGCGAAGGTCAACGGAGAGATGCTTCAGGTAGCAAACTACTAT

GACGTTGAGGGAAACCTTGTGGGTCAGAAGGTTCGCGACAAGAATAAAGAGTTCTCTGCAAAGGGTAAACTCAAAGC

TGACCTATTGTTCGGTAAGCAGTTATGGAATGGTGGCAAGAAGATTGTAGTTACAGAAGGTGAGATTGATTGCTTGAC

TGTAGCTCAACTTCAAGAAGGGAAATATCCCGTTGTGAGTCTCCCTATGGGTGCTCAAGCCGCAAAGAAAACATGCTC

TGCCAACTATGAATACTTTGACCAGTTCGACGAGATTATCCTTATGTTCGATATGGATGAACCCGGGCGTAAGGCTAT

CGAAGAGTGTGCTCCTGTCCTTCCTAGCGGGAAAGTCAGAGTAGCTGTACTGCCTCTTAAGGATGCCAATGAGTGTTT

ACTCAATGGACAAGCCAAAGCAGTAACCGACCAGATTTGGAATGCACAACCTTGGGTGCCTGATGGTGTAGTCTCTGC

TGTGTCTCTTAAAGACCGAGTGCGTGAAGCTATGGTCAAAGAGGAAACAACAGGTCTTCTATTTACGGGTCAACCGAC

ACTAAATGATATGACACTGGGTGCTCGTGGTGGTGAAGTCATCATGGTCACTTCAGGTTCCGGTATGGGTAAGTCAAC

GTTCGTTCGTCAACAGATGCTCCAATGGGGCAAGGGTGGTGCGAAGGTAGGCTTGGCTATGCTTGAAGAAGCCGTTGA

GGAAACTGTTCAGGACTTAATGGGCTTGAACAATCATGTCCGTCTACGTCAAGACAAAGAACTCAAGATGAAAATCTT

AGAGGATGGACGCTTCGATGAATGGTATGGTGCACTGTTTAACACAGATATGTTTCACCTGTATGATTCCTTTGCGGA

GTCACAGGAAGACCGTCTGTTTGCGAAGCTGGCCTACATGGTCGATGGCTTAGACTGTAACGTCATCCTGTTAGACCA

TATCTCTATTGTGGTATCTGGGATGGAAGATAACTCTGATGAGCGTAAGACGATTGACCGCTTGATGACCAAGCTGAA

AGCCTTTGCGAAGACTAAAGGTGTGGTCGTTGTGGTAATCTGTCACCTCAAGAATCCTGAGAAGGGGAAAGCACATG

AAGAAGGTCGTCCAGTTAGCATTACTGACCTACGTGGCAGCGGTTCTCTGCGCCAACTATCTGATACTATCATTGCTCT

CGAACGCAACCAGCAAGGGGATTACCCAAACCTTGTTCAACTTAGGGTGCTTAAGTGTCGCTTTACTGGTGATACTGG

TGTGGCTGGGCATATGGCCTACAACAAGGAAACCGGATGGCTTGAACCGACTGTCTCACCTGAAGATGAAGGAAGCA

GAGATAGCGGCTGGGAACCAGAAGAAGACGGTCAGGACTTCTGATAGTAAACCTATACAACTCACTAATGGGGCAGA

CAGTCTGTTCCCACTTATCAATCCACTTAAGGAGAATCACAATGTTTAACTTCATCAACACTCTTGGTAAGCTGGTAGT

TAAGCTGTACTTCTGTGAAGCTAAACGTATTAACAACAAGGCTCGTAAAGATGCTGAGCGGGCACAGCGCTTGGCTAA

ACATGCACGTATGCTGTCTGAAGATGCAAGTGCTGGTGTAACGTCTGCTGCTAAGATTGCAGCTATAGCAACCGACCT

CAACAAATTCTTCCTGTAAGGAGTAACATATCATGGCTATTAAATTCCCTGGCAATACATTCGTCTGTCTGACACAGT

TGACCAGTATGCTCGTCGAGTTCACGTCAACGTTCGTAACGGAAAAGTCACTCTGGTCTACCGTTGGAAAGACCATAA

GTCCACCAAGGCGCACACACAGCGTGTAACTTTGGATGACATGCAGGCAGGTCGTCTATTATCTTCTGTTGCTGTAGC

GGCTACTGTGGCTGTAGGTGAGGACAATGTACGTCAGGCTATGTTAGGCAACACAATTGAAGCTGAAGGCAACATGC

TTGCTGAGAAGTGTTAGGAGTAATAAGAGTTAAACTAAAGGTCATCTCCGGGTGGCCTTCGTGATTAACTTTTGTATG

AAAACTATTCAACTAACTAATGGAGTAAACACAATGTTACTATCCGACTTAGAAGCAAATGGGTTGCTGCACAGCGTT

AGTCAATTTCACTGTGCGGTGACTTACGACTACACAACTGACCAGTACATCCGTTATCGACCTGATGACTTTGGGGCAT

ACATTGATGCTCTCGAAGATGAGGTTGCTCGTGGGTGGTCTCATTGTATTTCACAATGGTCACAAGTATGACATCCCAGT

AATCGAGTTACTCGCCAAGAAACTGTTAGGACGTGACGTCGAGTTCCCTAAAGAGAACGTCTTGGATACCCTAGTGAT

GTCCCGCTTGGTTTACTCTAACATCAAAGACACCGATGCAGGTCTGCTGCGTGCTGGTAAGCTACCGGGTAAGCGCTT

TGGGTCCCACGCTCTGGAGGCTTGGGGTTATCGCTTAGGCGAAATGAAGGGTGAGTACAAGGACGACTTCAAGGCTTC

ACTGGTTGAGTTCTGGTGAAGACTACGTTGACGGTATGGAATGGTTGATGTTCAACGAAGACATGATGGAATATAACGT

TCAAGACGTTGTAGTAACCAAAGCTCTCTTCGAGAAACTTTGTAGTAACACATTCTATTTCCCTGAGGTGGAACCTAAT

GGAAGCACTGAGGCCGAACGTTTCTGGAAGGGTAGCATTGAGGCTGTCAAGCTGGAACACGATGCAGCATGGTTGTT

AGCTAAGATGGAACGTAATGGTTTCCCTATTGACGTCAAGTCCTTAGAGAATCTTTATGCTGAACTTGCTGGTCGCCGT

GGTGAACTTCTGGTCGAACTGACCAACACTTTCGGCTCATGGTATCAGGTCAAGGGTGGAGTTGAAGCATTCCGTCAC

CCAAGAACTGGTAAGCCTCTCACTAAGTATCCTCGTGTGAAATATCCGAAACAGGGTGGTATCTTTAAGAAGCCAAAG

AACAAGAAGCAACGCGAAGGTCTAGAACCTTGTGAACTGGACACTCGTGATTACGTAGAGGGCGCTCCATATACGCC

TGTAGAGCACGTTGTGTTTAACCCTTCTAGTCGTGACCATATCACTCGTGTGCTTACTAAAGCTGGCTGGGTGCCCGTT

GAGTTCACTCCTAGTGGTGCACCAAAGGTAGACGATGAGGTCTTAGAGCACGTTAAGGTTGATGACCCGGATGCACA

GCGTTGCATTGAGTTGATTAAAGAATACCTTATGATTCAGAAGCGTATCGGTCAGGTTGCTGAAGGTGATAACGGCTG

GCTCCGAATGATTGGAGAGGATGGTCGTATTCACGGTAGCGTTAACCCGAACGGTGCAGTAACTGGACGTGCAACTCA

TAGTTTCCCTAACGTTGCCCAGGTGCCTTCTATTCGTGCTGCTTATGGTGAACCTTGCCGTGCTGCTTTCGGTGCCGAGC

ATAACCTGAAAGACGGTAAGCCAGACCCTTGGATTCAAGTTGGTGTGGATGCGTCCGGCCTTGAGCTACGTTGCTTGG

GCCACTTCATGTATCGCTATGATGAAGGTGAGTACGTTGAGACTATCCTTACTGGTGACATCCATACGAAGAACCAAA

TGGCCGCTGGGTTACCTACAAGAGACAACGCTAAGACGTTTAACACAATGGGCGTCTATAAACCTATCTAATTCGATG

GACATCCCTATGGGACAATATCGAGCTAAATGATATTCGGTAGCCTTTAACTAAGGAGGCTCCATATGTATTCACTAA

ACCATAACAAACCTAAATCCGCACAGACCGCGACTCCCAGCAAGTACCCTCAGGGGCACTTTAAGGATAAGAAGTGT

AAGTGCTGCAATTTGGTATTTAAACCCAAGTCTCCTGCTGAGTTATACTGCTCAGATAGGTGTAAGGATAGGGGAATC

CAGACGGCATATCTTATGCCGGAGCTACGGGATAACTCTTGATGACTACGAAGAGATGTTGGAATCCCAGAAAGGTCTA

TGCAAGATATGTGGCACCGAGGGTTTTGCAATGAACCCAAAAGTGCACAAGATAAAACTTGTTGTTGACCACTGTCAC

ACCAAAGGTAATGTTAGAGGTTTTATTGTGTCACAATTGCAATCGTGCATTAGGGCTTCTTAAAGATAACGTGAAGTCC

CTACAGGCTGCTATTGAATATCTAAATGTGTAACGACTATCCCGGAAGGGAGTAGGGCCAAGTGGCTCGAAACGGTA

GGGCGCTGAGGCGCAAGATATAGTCTGCTCTGCATGGCAACATGTAGCAGTTCATAAGAGAACGGGTAGGGATTAAC

GACCCCTGTTGAACACTAAGGTATCTACGGATTTTTATATGGTGCTGGTGCTGCGAAGATTGGACAGATTGTTGGTGGT

ACTGCCGAAGATGGTAAGCGCCTCATCAAGAACTTCCTTGAGCAAACTCCGGCTATCGCTGCGTTACGTGAAGCAATC

ACAGGGACACTCGTCAAGGACTCTAAGTGGGTTGGTGGTGAACAGAAAGTAACTTGGAAACGCCGTTGGGTTCGAGG

CTTGGATGGTCGTAAAGTACACGTCCGTAGTCCTCACGCTGCGTTGAACACCTTGTTGCAGTCTGCTGGTGCACTTATT

TGTAAACTGTGGATTGTCAAGACAGAAGAGATGCTCCTTGAGATGGGCTACAAGCATGGCTGGGATGGTGACTTCTGC

CTCATGGGGTGGATACATGACGAGCAACAGCTTGCTTGCCGTACAGAAGAGATTGCCAAAGTTGTAATCGAAGTGTCT

CAACAGGCTATGCACTGGGTAGGCGACCACTTTAACTTCCGGTGTCAGTTAGATACTGAAGGAAAGATGGGGCCAAA

TTGGGCTGTGTGTCACTAATTAACAGGAGAAAAATATCATGGCTATTACTAAACGTTTTAAAGTATCATTCGAAATGA

CAGTTAAGGTTGACTCTGAGACTGAAGATAGGATTCAGGAACATATCTTAGATTTGGCTCGCAGCGTGTGTGATGGCG

AGAAAGTTAGTGCACAAGACAAAGAGTTACTCGTTCAGGCTCTGACATATGGTGTCGATGGTGCTGTGACATTCGCTG

TTAAGCAAGTTGTCCGTAATGAAATCCGTGAGATGGGCATTGAGCACCACGGAGAGGGCATTAAGTTTTCTCCAGCAA

CTATTCGTGAGGTGAAGTAATGGGTGAATATATACGTGTACTGGCGGCTATCAAAAGCTGCCCTAAGACCTTTCAGTC

AAACTACGTGCGTAACAATGCGTCACTGGTAGCTGAGGCTGCAAGCCGTGGTCATATCTCTTGCCTATCAGTCGATGG

GCGTAATGCTGGTGCATGGGAAGTAACAGGTGCTGGTGTCCGTTTCCTGAAGCAGATGGGAGGTTGTGTATGAGTTTG

CTTACTGACAACGTACAGGTGACTCGTGAAGCGTGGAATGACATGCAGGTGTACATTCAGTCCCTTGAGAAAGACCAA

GAGTTCCTGGATGCTCTGAGAGCCTGTGGTGTGGACAATTGGGATGGCTACCTAGATGCTGTCGAAATGGTGTGTAGG

GAGGATGACTTATGAGTGCCATTACACTGAAGGAGTTCTATGAGTTACGCCAAGGTAAATCTGATAAGGGAGTCTTGG

TGATGGATGGCGACTGGCTTGTCTTTCAGGCAATGGCAGCAAGCGAAGTCGAGACCCAATGGGATGATGACATTTGG

AGTTTAGAGTGTGACCACGGGAAAGCGTGGAACATTCTGGTAGACTCCATCAAGTCCTATGCTGGTCGCAAGAAGGCT

TGGCGTGATGCACCTATCGTGATGGCCTTCACTGACTCTGTGAACTGGCGTAAGGAATTAGTTGACCCAACCTATAAG

GAGAACCGTAAGGCGTCTCGTAAGCCTGTAGGCTACCGTGCGTTCGTCGAGCGTGTTCAAGCCTTTGAGGACTGGACG

TCTATCCTTGAGCCTCGCTTAGAAGGTGATGACGTAATGGGAATCATTGGGTCTGGTGCTGAACACTTTGGGTTCAAG

AAGGCCGTACTGGTCTCCTGTGATAAGGACTTTAAGACCATCCCTAATTGTGACTTCTTGTGGTGCACTACTGGTAACA

TCTTGACTCAAGACCAGACTACCGCTGACTACTGGCACATCTATCAGACCATTAAAGGTGACTTGACAGATGGCTATG

GTGGCATCCCAGGCTGGGGTGAGACAACTGCTGACTGGTTAGAGAACCCGTATGCCTTTGAACAGGTTGAGAAAACCT

TTAAGTCTGGTGCTCGTAAGGGACAGACAGTTCTCGAATGGAAGAAGGTGGACATGGGAGACAGAACCTTGTGGGAA

GCTATTGTGACCCTTGGTGCCAAAGCTGGGATGACTGAAGAGGAAATCATTAAGCAAGGTCAGATGGCTCGCATCCTT

CGTTTCGAAGGTACAACTTTATCGACAAGGAGATTTCCCTATGGTCTCCTGAACAGTTGCATTCGTGAGGGCTACACT

TATAGCTAAGAGTGTGTAACTCTAGGCATTCTTCTCTATGTGAAATTAATACGACTCACTATTGGAGAGGGGGTGCCT

ATATGATAACCTACTTTAAGTTCTAACTTTAAGGAGCACATTAATTATGTTAACCCCTATCAAGAAGTACATGGAGAAC

CCACAAGATATTCCTAATGTTCCTCGGAGTGTCATGGAGTATCTTCAAGTTCAATACAATGCTGGATACGCTATTCAGT

CTGGTCTTATCAATCGGTTGAAACAGGCTGGGTGGTCTGAAAGCTACATCGCTGGGTTCCTCGCGGGTCTCAACTATG

CATCTCAAACCTTAGACGATATGGAGGCAATCCGTAAGGAACAGGCAGAATCCTAATAACAGGAGGTAATCTGATGT

GTTTTTCACCAAAGATTAAAGTGCCACAGGTAGATACCAATCAGGTACGTGCAATTGACCCTGCTCCACTGACAGAGA

AACCTAAAGGTGTTCTGTTTGGTGGTGATGATACTGATGCACCAAAAGATGGTACAAGTTCTGAAGTGTCTACAGGTG

GTAGGAAGTCTCTCAAGGTGAAACTTGAAGACACTCCATCCAAGACAATGAAGGATGACAAGACAAAGAGACGAACC

GGGACTAAAGCATCTATCCGCAAGTCCGTATTCAACAACAAGTAACTAGGAGGTTTATCATGGGTTTGGGTAAAAGCT

TAAAGAAAGCTATCAAGAAAGTAACTAAGCCAATCGAAGGTATCACTAAAGGTGCCCTGGGAGTTGCTACTGGTCTA

CTGGGTGGTGGGTATGCCAGATGTACCACAAGCTGCACAGATTGTTGAGCCACCAAGTAAAGGTGACGTGGACACTGA

TGACGGTTCTCAGACTGAAAGTGGCAAGAAGAAGACTCGTGCTAGTGGTAAGAAATCACTAAGTGTCGCTCGCAGTTC

TGGCGGTGGCCTAAATATCTAACAAGGAGGCGCTAAATGGCAAGCTCACAGAAACGTGAAGGCTTCGCTGAGAATGG

TGCTAAGGCGGTGTATGACGCATTGAAGAACGACCGGAACTCCTATGAGACCCGTGCGGAGAACTGTGCGAAGTACA

CCATCCCCTCGTTGTTCCCTAAAGACTCCGATAACGCCTCTACTGACTACACAACTCCGTGGCAAGCAGTGGGCGCTC

GTGGTCTGAACAACTTGGCCTCTAAGCTCATGCTTGCTCTGTTCCCTATGCAGACTTGGAATAAGCTGACCATCTCTGA

GTTCGAAGCGAAACAATTGGTAGCACAGCCTGCCGAACTGGCGAAGGTCGAAGAAGGTCTCTCTATGGTCGAGCGCA

TCTTGATGAACTACATCGAGTCAAACTCATACCGTGTGACACTCTTTGAGACCCTCAAGCAGTTAGTCGTAGCGGGTA

ACGCTCTGCTATACATTCCTGAACCTGAAGGTACTTACAACCCCATGAAGTTATACCGGCTGTCTTCTTATGTTGTCCA

ACGAGACGCATTCGGCACGGTACTACAGATTGTGACCTTGGATAAGACTGCCTATGCAGCACTTCCAGAAGACGTAAG

GAACTCTATGGACTCTAGTCAGGAGCATAAGGGTGACGAGATGATTGATGTGTACACTCACATATACCTCGATGAGGA

ATCTGGAGAGTACCTGAAGTACGAAGAGATTGATGGTGTTGAAGTCGAAGGCACAGATGCTTCTTATCCGGTGGACGC

TTGTCCATATATCCCGGTTCGCATGGTGCGCATTGATGGAGAGTCATATGGTCGTTCGTACTGTGAGGAATACTTAGGT

GACTTACGTTCACTTGAGAACCTCCAGGAAGCAATCGTCAAGATGTCCATGATTAGTGCAAAGGTAATCGGCTTGGTT

AATCCAGCTGGTATCACACAGGTTCGTCGCTTAACGAAAGCACAGACAGGTGACTTTGTGTCTGGTCGTCCAGAAGAC

ATCTCGTTCCTTCAGTTAGAAAAGGCTGCTGACTTCTCTGTAGCGAGAGCTGTGAGTGAACAGATTGAGGGCCGACTG

TCATATGCCTTTATGTTGAACTCTGCGGTACAGCGTACTGGTGAGCGTGTGACAGCCGAAGAGATTCGCTATGTTGCAT

CTGAACTGGAAGATACCCTTGGTGGTGTCTATTCGATTCTCTCACAGGAACTCCAGTTACCTATGGTTCGTGTACTACT

GAAACAACTTCAAGCAACCAATCAGATTCCTGAGTTACCTAAAGAGGCCGTTGAGCCAACTATTAGTACCGGGATGG

AAGCATTGGGGCGTGGCCAAGACCTCGATAAGCTGGAGCGGTGTATATCTGCATGGTCTGCTCTGGCTCCTATGCAGG

GAGACCCGGACATCAACATTGCGACCATTAAGTTGCGTATTGCGAACGCTATTGGCATCGACACTTCTGGTATCCTTA

AGACACCTGAAGAGAAGCAACAGGAAATGGCTGAGGCCGCCCAAGGTACTGCTATGCAGAATGCCGCTGCGTCTGCT

GGTTCTGGTGCTGGTGCAATGGCTACTGCAAGTCCTGAGAACATGGAAGCTGCTGCTGCACAGGCCGGTATGGTTCCT

AATTAATACAACTCACTAATGGGAGAGACACTATGTCCTCCCTTTGAGTTCTACAATTTATTATCAAAGGAGATACAA

T ATGTCCGGTGAATCTAATGCTG ATGTTTATGCATCTTTCGGTGTGAACTCTGCGGTTGTCGGTGGGTCTACTCCTACT

GACCACGAACAGGCTATGCTTGAACTCAATGTTGCTGCCCGTGATGGCGATGATGCAATCGAGATAGTAGGCAATGAT

GACCCTTATGGTAATCCAGACCCGTTT GGTGAAGAAGAAGACGAGAAT CGTACTGAGATTCGTATCTCCACTGATGGT

GAACCTGAAGAGACTGAAGAGGGTGAGATTGATTACTCTGAGAGCACTGAAGGAGGCTCAGAAGAGTTTTGCACCTGT

AGGTGATGTTCCAGAAGAACTAAGTGCAGCCTCAAGTCAACTCGAAGAGCATGAGACGGGCTTCGAAGAGATGGTCA

ACCAGGCTGCTGAACGTGGTCTGTCTGAAGAAACTATTGTCCGTATCCAACAGGAATATGGTGAAGAGGGTCTGTCCA

AGGAATCCTATGAAGAACTGGCTAAGGCTGGTTACTCTAAGTCCTTCGTTGACTCCTACATTCGTGGACAGGAAGCAC

TGGTCGAACAGTACGTTCAGTCTGTTATGGAGTATGCTGGTGGTGAGTCACAGTTCCAGGCAATCTATAGCCACCTCG

AAGTGTCCAACCCGGATGCTGCCGAGTCTCTGGTTAATGCTCTGGAAAACCGT GACCTAGCGACCGTTA AG GCTATCG

TGAATCTTGCTGGG GCAAGCCGTACTAAGACTTTTGG TAAACCTGCTGCCCGTAGTATTTCTAAACGTGCCGTTCCTGC

TGCACCTGTACGAGCCAAACAGTCTGGCTTTGAGTCACAGGCTGAGATGATTGCTGCAATGTCTGACCCTCGCTACCG

CTCGGACTCTAAGTTCCGTGCTGAAGTTGAGCGTAAGATGATGTACAGTAACTTCTAAAATTAATACGACTCACTATT

GGGAGAGACATCACGTTCTCCCACTTTGAGTGATACACAATGAGAACCAACTCGTTTCCAAGTAGTACCTCACAAATTA

AAACTTTATAAACCAACAGAAGGAGATTCAACATGGCTAACATGAATGGTGGACAACAGATGGGTAAAGACCAAGGT

AAAGGCATGAGCGCTGGGGACAAACTGGCTCTGTTCCTGAAAGTCTTTGGTGGTGAAGTTCTGACTGCGTTTACCGT

ACCTCCGTAACTATGAACAAACACCTTGTTCGCTCTATCCAATCTGGTAAGTCTGCGCAGTTCCCTGTGCTGGGTCGTA

CAAAAGCTGCTTACCTGCAGCCAGGTGAGAACCTCGATGACAAACGTAAAGACATGAAGCACACTGAGAAGACCATT

AAATTGATGGGTTACTTACTGCTGACGTTCTGATTTACGACATCGAAGACGCTATGAACCACTATGACGTTCGCTCTG

AGTACACTGCACAGTTGGGTGAATCTCTGGCAATGGCCGCTGATGGTGCTGTACTGGCTGAAATGGCTAAGTTATGTA

ACCTTCCAGCTGCTAACAATGAGAACATTGCTGGTCTTGGTAAGGCTCACGTACTTGAAGTTGGCGACCAGGCTACCC

TGCAAGGTGACCAAGTTAAGTTAGGTCAGGCGATTATTGCACAGTTGACTCTGGCTCGTGCTAAACTGACTGCCAACT

ATGTTCCATCTAGTGACCGTGTGTCTACACCACTCCTGATAACTACTCTGCGATTCTGGCTGCTCTGATGCCTAACGC

TGCAAACTATCAAGCTCTGATCGACCCATCTACTGGCTCTATCCGCAACGTAATGGGCTTTGAAGTAATCGAAGTTCC

ACATCTGACTGCTGGTGGTGCTGGAGATAACCGTGCTGAAGAAGGTGTAGCACCTACTAATCAGAAACACGCATTCCC

TGATACTGCTTCTGGTGACACTCGTGTTGCTTTGGATAACGTGGTTGGTCTATTCAATCACCGCTCTGCCGTTGGTACT

GTTAAGCTGAAAGATATGGCTCTGGAACGTGCACGTCGTGCTAACTTCCAAGCAGACCAGATTATCGCTAAGTACGCA

ATGGGTCACGGTGGTCTACGCCCTGAAGCATGTGGTGCGCTGGTTTTCAAAAAGGCTTAACTTCTCGCAAACTCGTAG

GGGACACTGAGTCCCTTACTGAAGTTTAGCAAGAGCTAACGCCGCAACAGAAAGCTGCCCGTACCCGTGCGGCAAAC

AAGGCTCGTAAAGAAGCTGAGTTAGCTGCTACTAAGTAACAACTATATGAAACCCCTTGGGGTGCCTTCGCGGGTGCTT

GAGGGGTTTTTTTGCTTATGAGGAGGATACACTTATGCGTTCCTATGAAACTACCCTAGAGACAGGCGAAGAGTTGGC

TGCTGTCAACGACATCTTGGCCTCTATTGGTGAACCCCCAGTATCCACTCTGGAAGGTGACACTAATGCTGATGTTACT

AACGCCCGTCGAGTTCTCAATAAGATTAACCGACAGATTCAATCGAAAGGCTGGGACGTTTAACATTGAGGGAGGACA

ACAGTTGCTACCTGATTGTGTTCAACGGGTTTGATTCCTTACATGTCAGACTACCTGTCTGTACTCTCAGAAGGTGGTGCA

ACTGCCTACGTTAACCGTGGTGGATATGTGTTTGACCGGACGACCGGAACTGATATCTTTGAGGGCCCAGTTACTGTG

ACCATCATCAAACTCCGAGAGTTCTATGAGATGCCTGAATGCTTCCGCTCATGGATTGTCACCAAGGCAGCCCGTCAG

TTTAACAACAGGTTCTTTGGTGCACCTGAGATTGACGCAGTACTAGCTGAAGAGGAACAGGAAGCCAAGATGCAGTG

TCACGAGTATGAACTGGACTTTGGTAACTTCAACATGCTTGATGGTGATGCCTTCACAGGTGGTCTACTATCTCGTTAA

CGCTAACATTTTAAAGGAGGTCTCTAATGGCTCTTATTTCACAATCCGTCAAGAACCTGAAGGGCGGTATCAGTCAAC

AGCCGGACATCTTAAGGTTCCCCGAACAGGGTCCGAACAGATTAACGGTTGGTCCTCGGAGACTGAGGGTCTTCAGA

AGCGTCCACCTTTTGTCTTCACTAAGACCATTGGAGACCAGAATGCCCTTGGTGCCAAACCTCTAGTTCACCTCATCAA

CCGTGATAGTGCCGAACAGTATTACGTAGTGTTTACCGGACAGGGTGTTCGTGTGTTCGACCTAGATGGTAAAGAGTA

TTCTGTGAAGGGTGATTTGTCCTACGTTAAGGTAGGGAACCCTCGAGATGACTTAAGGATGGTCACTGTGGCTGACTA

CACATTTATCGTTAATCGCAATATGGTTGTGCGCCCAGATACTACCCCTTTGTATAACCCTTAAGGAGAATGGAGACTGC

TTGATTAACATCCGCGGGGGTATGTATGGTCGTACGTTGGCATTCACTATTAACAACACAAAGATTGCATACGAGATT

GCCCATGGGGACGTACCAGAACATTCTAAGCAGACTGACGCACAGTGGCTCGTTAAGAAACTTGCTGGTCTCGCGCGT

TTGAATGTTGCCTTTAAGGGCTGGACGTTCACCGAAGGGCCTGGGTACATCCATGTGATTGCTCCAAGTAACAGTCAG

ATTAACTCACTGTCCACTGAAGATGGCTACGCCGACCAGTTGATGAACGCAGTGATGCACACCAGCCAGTCATTCAGT

AGGTTGCCCGTTGAGGCTCCTAATGGATACACGGTGAAGATTGTTGGTGACACCTCTAAGACATCCGATATGTTCTAC

GTTCAGTACGACAACTTGAAGAAGGTGTGGAAAGAGGTTGCAGGTTGGGGCGTACAGAAGGGACTTAATGGTGACAC

GATGCCTCATGCTCTTGTACGTCAGGCTGATGGATCATTCCAGATGCAAGCACTACCGTGGGCGCAACGTACCTGTGG

TGACATGGACACTAACCCTACTCCGTCTATTGTTGACCAGACGATTAACGATGTGTTCTTCTTTCGTAACCGCTTAGGG

TTCCTCGCTGGTGAGAACATTGTGATGTCCCGTACCTCTAAGTATTTCTCATTGTTCCCTGCATCAGTAGCTAACCTGTC

CGACGATGACCCTATCGACGTGGCTGTGTCTCATAACCGAATCTCTATCCTGAAGTACGCTGTGCCGTTCTCTGAGGAG

TTGCTCCTATGGTCAGACCAAGCACAGTTCGTGTTGTCCGCTCAAGGTATACTCTCACCTAAGTCAGTAGAGTTGAACC

TCACGACCGAGTTCGATGTGTCAGACCGAGCGAGGCCTTTTGGCGTTGGACGTGGTGTGTACTTTGCGTCACCTCGTGC

TTCCTATACGTCACTTAACCGTTATTATGCGGTACAGGATGTTAGCTCTGTGAAGTCTGCTGAGGATATGAGTGCTCAC

GTTCCTAGCTACATTCCGAACGGTGTGTTCTCTATACGAGGCTCCAGCACTGAGAACTTCATCTCTGTGTTGTCTTCAA

ATGCTCCAAGCAGAATCTTCCTCTATAAGTTCCTCTACTTGAACGAAGAGATAGCACAACAGTCGTGGTCACATTGGG

AACTAGGAAGTAACGTTACCGTACTGGCCTGTGACTCTATTGGTTCCACCATGTACCTTGTGTTGCGCAATCAGTCACA

CACTTGGATGTGTCGTGCTCACTTTACGAAGAACAGCATCGACTTCCCTGATGAGCCGTATCGTCTGTACATTGACAAT

AAGGTGAAGTATGTGATTCCTGAAGGTTCATACAATGATGACACTTACGCTACCACTGTGAAACCTGTAGACGTCTAT

GGGATGAAATACTGGACCGGTAAGTTCTACATTGTGGCCTCTGATGGACTGGTGTCATGGTTCGAGCCTCCTCGTGGT

GGTTGGCCTAATGGTGTACCTATGTTAACAATGAGTGGGAACCGTGAGGGTGAGACAATCTACGTTGGGTTGGCTATC

AGCTTCCGCTATGTGTTCTCTAAGTTCCTCATTAAGAAGACCGCTGATGATGGCTCTATAGCTACCGAGGACATTGGTC

GTTTACAGTTACGCCGAGCATGGGTGAACTACGAGGACTCTGGTGCATTCACAGTGGAAGTGGAGAACACCTCTCGAC

TGTTCAGCTATGATATGGCAGGTGCTCGCTTGGGTTCCAATGCGTTACGTGCTGGTGGACTTAATGTTGGTACGGGTCA

GTTCCGCTTCCCGGTGACTGGTAATGCACAGTTGAATGAAGTACGTATTATCTCTGAACACACCACACCACTGAACGT

TATCGGTTGTGGTTGGGAGGGTAACTACCTTCGTCGTTCTTCTGGTATCTAATTGAAACACACCCTCTTACTCATTCAG

TGAACAATACGACTCACTATTGGGGAAGGGGGTTTATACTATAGGAGAGAAACTCTATGATTATCCTACCTACAACAG

AACGTGACTTTATGTTATTCAAACCATCAGAAGAGGACATTGCTGAGGCCAAAGCCTACGGGATTGAACCTTCATTTC

CACCAGCCTCTGAGTGTGTGACCATGAGTTTACATGGGGTTCCTTTAGCTATTGGAGGCAACTGTGGTGACCAAGTGT

GGTTCGTAACGTCTGACGAAGTGTGGAAACTATCGAGACCTGCTAAACGGGAGTTCCGCAAGCTCATCCTAGAGTACC

GCGATACGATGCTAAAACAGTATCCAGTAATCTGGAACTATGTCTGGATAGGCAATAAGTCACACATTAGGTTCTTGA

AGTCTATCGGTGCGGTATTCCACAATGAGTTTACTGGTGACAGTAATCAATTCCAACTATTCACAATAGGAGGCTAAA

TTATGTGCTGGATGGCGGCTATCCCTCTCGCTATGCAAGGTACTTCCATGATTATGGGAGGCATGCAAGGTGAACTAG

CAAAGGCTGCACAGATTGACCAAGGTCGTCGACAAAGTTGGCAGATGCTGAAAGAGATGAATTATACGGATGCTAAC

CTTAAGTTAGAATCTCGTGACCTCATCGACTCAACTGTCCAAGAGATGACCCAAGCCAATATGAACCGTGTGAGAAAC

ATGGGGACTATCCGTGCGGCTATCGGTGAAGGAATGCTTGAAGGTAACTCTATGGACCGCGTACAACGCGTGACCGA

GGGTGACTTCCTTCGAGAGTCTCAAGGTATCACCGAGAACTACCAACGTGACTATAGTGTCATCTTAGGGAAGCGTTT

AGCTAACCAGGAGAACACCGTTAGTCAGATAAAAGAAATCAACAAGTCCGAGCCGAAACTAAAAGGTCGCTTAGAGC

AAATCATCGATCCACTAGGTATGGGTATGGGTCAACTAGCTGGTATCGCAACTTCTGGTGGACTCTTCGATAGCAAGT

CGACTAAGAAGGCTACCACTAAGGTTAAGGCCCCGGGCGTTAAGTGAGCAGATCAAGAAGGTAAATAAAGGAGGTAC

AGTTTATGAGTAAGTTAGCGCAAGCATTAGGTAGCATGAACGCTCCGTCTACTAGCCGTCTGCGTGGCACTGGTCGCA

TGGAGGTTAAGGCTGCAACAGTCTATGAAGACCCTAAGTATGCCGAGAAGTCTAAGCTGATTGGCACTGTAGGTAAA

CTGGCTGAGATGGGTGCGGATGCCTATATGCAGTACGACCAGCGACAGAAGGACAAGGCAGACGAACGCTCTAACGA

GATTATTCGTAAGCTGACTCCTGAGCAACGCAGAGAGGCCATTAAGAACGGAACCCTGTTATATCAGGATGACCCATA

CGCAATGGAAGCACTCAAGCTTAAGACTGGACGTAACGCTGCGTATCTCGTCGATGACGAAGTGGCACAGAAGGTGA

AGAACGGTGAGTTCCGCACTCGGCAAGAGTTGGAAGAGTTCCGTCACAGTCGACTGCAAGAAGCCTCTAAGAACTAT

GCGGAACAGTTTGGTATCAATGAGACTGATGAGTTCTACCAGAAGGGTTTCAACTCCGACATCACTGAACGTAACATT

GCCCTCTATGGTGCTCACGATAACTTCCTGAGTGACCAAGCTAAGAAAGGTGCTGTCATCAACAGTCGCGTAGAGTTA

AACTCTGTGTTGAACGACCCGGAAACCCTACGTTTCTCCGTATGCTGGTGAGTTCTTTGAGAACTACTTCAGTGCCGGAT

TGACGACTGGGAGTATCCCTAGTGATGACCAAGCGTTCACAATGATTAGCCAAGGTCTGTCTGACGTGGTTAACCGTG

AAGGTGGCGGGCAGTTCCTGCAACAGATTGAGAACCGTAAGGTGAAACTCCACGGAAAAGAGACAACCTACAAGGA

ACTGATGGGCGCCGAACAGTGGAACAATCTGATGGTTAAGGCTCAACATAATGAGTTCCAGTTGAACGCTAAGAAGA

CCGAGGCGTCCAACTTAATGTGAACTCTGCGTTGAACCAAGAGAACATAAACACTGGTTGGGAACAGCTTCAATCTA

TCAAGGCTGAACTGGACACATTGCAACCGGGCGAAGAGATGACACCTGAACGTCAGGCGTTAATCAATGCACAGACT

CAGATGCAGGACAGGATGAATCGGGAGACCGCTGAGTTAGCCAAGCAGATGGACAAACAACAGAAGTCGATGAACA

AGATGAACGTCATTGATGCACAGTTCCAGAAGAGACTTAATGGTCAGTACGTGTCTACTGCGTATGGCGATATGCCTA

CCAACGATAACACTGGTGAGTTCACTCATAGTGACATGGTTAACTATGCGAACAAGAAGCTGGCTGATATTGATGCTA

TGAATATCCCTGATGCACAGAAAGACCGAATGAAGTTGGACTATCTCAAGGCTGACTCCGAGAAGGGTGCTTCCGTA

CTGCGGTTGGTGAACTTATTGGTGACCGCTGAGAAGGAATGGACGTCCGCTGTGATTAACGGGAAGATGCCCGAAGAC

GGTGGTGTGGCACTCAATGCCCTTCGACGTGTTCGTAATGCTGACCCCGATTTGTTCGCTGCATTATATCCTGATAAGG

CCGAGATGTTCCTCACTATGGATATGATGGACAACCAAGGGATTGACCCACAGATCTTACTGGATGCAGATAAGGCTC

GGCAGTCTCTAACCAAAGAGATGCAGTATGAGGATGATAAGTATTGGGCTTCTCTAATGAACAACTCGCAGTCCCCTG

AGATTAAGTACATGCCATCACTTCTCCAGAACGGTGCCCGTAAGATTTATGACTCTGTTAAGTACCGCACAGGTAACC

CTGATATGGCAATGCAACAGGTCGATAGGTATCTCAAAGATAACACTACGACTCTTACAGGTGATGATGTTGAAGGGG

ATACCATTGGTGTCTTAACGAGAAACTCTCTGCGTGTCACTGATGACCCCGGACTCTTGGAAACATGGTAAGGACATTA

TAGACACAGCCGCTAAGAAACTCGCTGAGACTAACCCTTGGGTGACTCGCAAGCAACTCACTGTGTTTGAACGTGGTG

ATTCTATCTACCTGATGGACACGACCGGACAGGTAAACGTTCGGTACGACAAGCAGTTGCTCTCTAAGATGTACCAAG

AGAACCAAGCGAAGCTGGATGAGGAAGCCCGTAATAAGGCACTCAAAGATGCCAACAAGCGAACCCTACATACACG

AGCTATGAACCGCAAGCGTGAACGTGAAGCTAAGAAGCCTAAACGCTCTGGCAGCATGTACGATAGCGTGAGTGGCA

AAGGTATTCTGGATACACTTACTGGTAAAGACTAATAGACCACGATAGGAGGTTCCAAGTGGATAAGTATAACCCGA

ACGAACCGCATGAATATGATGCGTTATTTCAACAAGCTGCTGACACACATGGAGTCTCCTATGGCCTTCTTCGGAAGG

TCGGTTGGTAGAGTCCCGCTTTAAGCCCACGGCTCAATCACCAACAGGTCCAAGAGGTGTCATGCAGTTCACTAAGG

CAACAGGTCAGGCTTATGGTCTCCAGAACGATGAAGACTTCACTGACCCTGCTAAGTCAATTGATGCAGGTGCTCGTT

ACCTTTCAGACCTCGTTAAGAAGTACAACGGGGATGAACTTAAAGCTGCCTTAGCGTACAACCAAGGTGAAGGACGT

AATGGTAAGCCTCAGTTAGATGCCTACGATTCTGGCAACTTTGCGAGCATTGGGGACGAAGGCCGTAACTATCTGCGT

TCACTGCTGGATGTTGCTAAGTCTCCTAAGAGTGGAGACCTTGAGTCATTCGGCGGTATCACCCCAAAGGCTAAAGGG

ATTCCGCTTGAAGCTGCTATGAGCGGTATAGGGAAGAAAGGAAGTGTGACAACTGAGCTCCCTGAGTCTCACGACAT

GTCCTTTCGAGGTAAAGAACAGGCCGTCCCCAATCAGCCGTTCGGAAAAGATTATTGGGAAGCCAAAGGTACAACTCT

TGACGAAGCAAACAAGCGTTCAACCTTCTTTGGATTCGGTAATGCTACTGAAGCAGAACTCTCCAACTCAACCTTGGG

TGTTGCCTTCCGTGCTGGTAAGCATGACGATGGTTTCGATGTTCTTACTGACGTACTTCAACCGACACGGGTTTAATAGC

CACATTTGGACGCCTGAAGAACTGGAGAAGATTAGGACTGAAGTGAAGAACCCTGCGTACATGAACGTTGTACTTGG

GGGTTCCGCTGAGAACCTTGACGAACTAATCAAGATGGCTAACGAGAACTATGAGACTGACGCAAAGGCTGCAGACG

CAGGCCTAGGTGCCAAGCTGTCCGCTGGTCTTATTGGTGCTGGTATTGACCCTCTGACCTATGTTCCAATCGCTGGGAC

TACCGCTAAGGGGTTCAAGTTGGTGAACAAAGCGTTAATGGTAGGTGCTCAAGCTGGTGCACTTAATGTTGCCTCTGA

GGGCCTACGTACTTCGATTGCTGGAGGTGAGGCCCACTATGCTGAAGCTGCCTTAGCTGGGATGCTACTTGCTGGTGG

ACTGACTGCTGTGGCAGATGGTGTAGCTGCTGGTCTCCGTAAGTCTGGTGCTGAACAGATTGAGAACCCGTTTGCTGC

TGCACAGATGCGCTTTGAGGCCCGTGAGACTGCACGCAACACTGGTGGTTATGACGCAAGCCGTATGCCTCCCTAGTGA

AGACCGAGTGTTCTCACAGCACAATGGCGTTGAGTATGCACCACTGGAAACCGAACCGGGTGCTGTAGTACTGAAGG

ATGGTTCCATTATTAGTGATACCAATCTAGCGAACCCTATGACAGCCAAAGAGTTTGCCGAGGTGGACCCTGAACGTG

CTGCTTGGGGATTGCCGATGCGTGGCCTGAGTGAAATAGGTTTGAGAACACTACGTTCCGAACATGCTGAAATCCGTG

GTCTCGCCAAGGACTTAGTGCGCTCACCTACAGGTATGGAGTCTGGCTCACACGGTAAGTTTGGGGCCACTGCCTCCG

ATATCAAAGAGCGACTGCACAGTACCAACCAGAGAACCTACAATGACCTCTATGGTGCAATGAAGGAAGCAATGGCT

GACCCTGAGTGGTCTGTGGGTGTGTTCAAGAGTGGTGCTCAAGGTGCTCGTCAAGAAATCTATCGTCGGGCAGCTGTG

GCTATCGAGCGTCCTGAACTTCAGGCTAACTTGACCAAGGCAGAGCGTAAGGTGATGGACATCATGAAGGAACACTTT

GACCTCAAGCGTGAGATGATGGAGAACCCGTCGATGTTCGGCAACAAGGCCAACTCTATATTCCCTAACAGTCGCCAC

AAAGGGACTTACGTTCCTCATGTGTACTCAAGGGAAGCCAAGCAGTTGTACTCACAGGCTCTGGGAGGTACCGATGGT

CTACAGGAAGCAATTGCTGCAAGCTGGATGACATCCTATCGTTCACGACCTGAAGTGAAAGCTCGTGTTGATGAACAC

TTAGCGGAAACCTTGGGTATTGACGTTAAGGCTGTTACTGAAGAGATGGTCATGAAACACGCTAGTGACAAGGCATAT

GGTATCGCCAAGACTGATCAGTTCAACTCTTCCAGTGTCCTTGATGATAACATTGAGGGTATTGCTGGTATCGAGAAC

AACTCATTCCTTGAGGCCCGTAACCTGTTCGACTCTGACATGCCTGTAACTCTCCCTAACGGACAACAGTTCTCCGTGA

ATGACTTACGTGACTTCGATATGAAAAGCGTCATGCCAGCCTATGACCGCCGTGTTGATGGTGATATTGCTATCATGG

GTGGTACAGGGAAGACTACTGCTGAACTCGTAGCTGAAATCAGTGCTCTCGATAAGAAGTTTGAAGGTAAGGGCACA

GTGAAGGGTGAGGTAGGAGCACTGAAAGATACCATTAAGATTCTCACTGGCCGTGCTCGGCGTAATCAGGATACTTTG

GGAGACACTATGGTTCGCGCTCTGTCCGATATGTCCTTCTTTACGAAGAACGCATACATGGGCTTGCAGAACCTAACT

GAAATCTCTGGTATGTTGGCAAAGGGTAACATCCGTGCGATGCTCCATGGTGTCCCAGCACTACGTGACTTAGCGTTC

CGCAACAAGCCTGTCTCTGGTAGTGAACTCAAAGAACTCCACTCAATGGTGTTCGGCAGAGAGTTCGACCAGTTAATT

CGACCGTCCCGTCAGGACATCATTCAGCGTCTCCGAGAGTCCACCGCTACATCAGATGTAGCCGCTAAGGTTGCTGGT

ACTATCAAGCATACGACTCAAGAGTTAGCTGCCCGCTCACCTCTAACCAAGTTCCTCAATGGTACGTCAAACTACCTTC

TGGATATGGCTCGACAGGGTGTGATGGGTGATGTGGTAACTCATGCTGTTACAGGCAAAGGTGCCAACAAGTGGATTA

AGGGTGACATGCTGAAGTCTGCGAGTATCTCTAAGGAACAGTGGGAAGGCATTCAGTCTCTCATTAGGGAGAACGTA

ACTCGTGGTGAAGACGGTAAGTTCACCTTCAAGGATAAGCGTAAGCTGGCTAATGATCCTCGTGCCATGGACTTATGG

CGTTTAGCTGATAAGGTTGCTGACGAAACGATGCTCCGACCTCATAAGGTTTCCTTACAGGATTCCCATGCGTTTGGTG

CCACCGCTAAGTTAGTCCTGCAGTTTAAGTCGTTTGTCATCAAGTCGATGAACTCTAAGTTTATCCGCTCTGGTAACGA

AGCCTTTAAGAACCATCGCGCTATGGACATGGCGTTGACCTATGCTATCTCTGGTGGTATTGCTGGCTCCTACTATGTG

GCACAGGCTCACTTGAAAGCTTGCTGGCCTACCTAAAGAGCAACAGAAGGAGTACTTGAAAGAAATCCCTCGACCCTAA

GATGATTGCCTACGCTGCGGCCTCCAGAAGTTCACACTTAGGGGCTCCGCTGAGTATTGCTAACTTCGGTATGGGAGC

TGTTGGTTATGACCAAGGTCTTATGGTCCGCTCTACGATTCTACCTAAAGGTGAGGACAAACGTGAGAGCAATAAGGC

AGTATCTTCTCGTGACATGGGTGATTCCATTATGGGCGCTATTGGTGAACAGGTGCCTGCTCTCGGATTCGCTGGTGCA

ACTTTTGCTGCTAGTCGGAATGCTTATGGTGTCCTTACAGCTCCGAACAAACTGACCGAGCGTGAGATGATGACTGGC

TTAATGAATGCTCACCGAGAGTTGATACCTAACGACCCAATCTCTCAACAGATGCTCATTAAGTTCTATGAGGCCAAC

GGTGTGCATATTAAGTCTAACAAGAAGTAAACAATACGACTCACTATTGGGGAGGCTCTCATAGCTCCCCCTTTTTATT

TACCCTTTATAACCTCACCAAAAGGAGGATACAGATGGCTAACAAAATTTCCACTGTACGTACTTATCCGCTGAACGGT

TCCGTGAACTTCACGATTACCTTTGAGTATCTAGCACGTAAGTTCGTCCTAGTGACACTTATAGGGAAAGACCGTAAG

GAGTTGGTTCTTAATCAGGACTACCGCTTTACCGCTAAGACCCAAATCACAACCGCCCGTGCATGGACTGCTGCGGAT

GGCTATGAGATGATTGAGATTCGTCGATTCACTTCTGCAACCGACCGTCTGGTTGACTTTGCTGATGGCTCAATCCTTC

GCGCATATGACCTTAACATCTCACAGATTCAGACGATACACGTAGCTGAGGAGGCACGAGACCTTACTGCGGATACTA

TTGGTGTCAACAATGATGGTAACCTAGATGCTCGTGGTCGTAAGATTGTGAACTTAGCGTTTGCTACTAGTGACTATGA

TGCTGTCCCTCTGAAGCAGATTACAGACCGTGAGTCCTCTGTGTGGAACGCTGTGACCAAAGCTAGTGAACAAGCTGA

CCGGTCTAACAAGGAAGCCAATCGCTCACGGGACGAAGCTGACCGCGCTAAACGTGAAGCTGACCGCTCGACCCAAC

AGGCAGGTGTCTCTGCGACACAAGCAGTTGAGGCCAAGAAGCAAGCTGACCGTTCCAACTCTGAGGCCAACCGCTCT

AAAGGTTATGCTGATAGTATGACAGCCAGTGTTGAAGCTGCTAAGGGTCACGCTGAGAGTGCCAGTAAGGAAGCTAA

TCGTTCCCGTGCTGAGGCTAATCGTGCTGCTGATGAAGTTACTAAGGCCGCTGCTGAGGTTTCCAAAGCTGCTGCACAT

GTGGCTAGTGCTAAGAATCAGGCCGACCGTTCCAGTACTGAGGCTAATCGTGCTAAGAGTGAAGCAGACCGTGCTAA

GACTGAGGCCGATAAGTTAGGCAATATCAATGAGTTCGCTGGAACTCTCGAGAAGGTGGAGGGAGTAACACCAACTT

TCAAATCGGGGATTAAACTTCGTTCTGGTGACTTCTATGCCGAGAGTGGTAGCTTCACTAAGAAAACACAGGGTGGCG

ATTGGAGCCAGTGGACTGGAGCTCTGACACCAGATGTACAACATGATATAGAAGGACAAGGGAACATCGCGTATCTC

CTTGAGCGAGCTACAACTTTGAGTAAAGAGCCCTTATATGCTAAGTGCCTATAATAATGGTGTTGGCGAAAACTTC

TTTATCAGGCAGTATCTTCGAGATGCACATTTTGACTTACGCGCCGATGGTGCTCTTATCAGCTCTTCTGGTTGGAGTA

TCCCAGCCGATGGTAACTTATACATCAAGAAGTATGGGTCGAATCTTGATACATGGGTTAATAAGCGCCTAAGTGCAC

ACGCCTACAGCAAGGGTGAAGTTAACAAGATGGTCGATGGACTACTCACTACCGAACAAGGTGATGCTCGTTATGCA

CGCAAGAGCTCTGGTTGGACTGAGGTATGGCAAGGCTTCTGCTGGTGGTGGTGTTTCAGTAAGCCTCTCACAGGATGTC

CGCTGGAGAACTATCTGGATTCTAGCTAATAATGGCATGTGTTCTGTTCAGATTGGAGCTGATGCTACTTACTTCATGG

TGGTTATGGGTGGTTGGTTGAAGTTCACAATTTCCAACAACGGGAGAACTTTCCGTAACGACCAAGATCGAAATACAG

TACCTGAGCAAATCTTAGTTCGAAACTGATAAGGACAATACACAATGACCATGGACCGGTCTTTACCAATGAGGAGGA

CACAATGTTGTCTTTAGACTTTAACAATGAAGTAGTGAAGGCTGCTCCGATTGTAGGGACAGGTGTGGCCGATGGTGC

TGCCCGTTCTGTTTTTCGGACTGTCGCTTAACGAGTGGTTTCTACGTGGCCGCAATTGCCTACACAGTGGCTCAAATTGGT

GCCAAGGTAGTCGATGTGATTATCAAATGGAAGAAGGAGGGTAGAGATGTCTAACGATAAGTCTTTAATACAATTCCT

TGAGATGCTGGACACTGAGATGGCACAACGTATGCTGAAAGACCTTCAGGATGATGAGAAGCGCACACCTCAACTGT

ACAACGCAATCGGGAAACTGTTAGAGCGCCATAAGTTCCAAGTCTCCAAGTTACAGCCAGACCGTAACATCCTTGGTG

GACTGGCAGATGGACTCGAAGAGTACCAGAACCTAGTGGGTGCTGATGGTCTCACTGAAGACGAGAAGTACACGCTG

AACTGATGGAATAACTCAAGGCCACTACATCTAGTGGTCTTTATGATATTTCAACACAACTCACGGTGACGCTACTGTG

AAATCTGAAAATACGGGAGGGTAGTTATGCTCAAGAATTTAAAGAGCTACGCCATAATCGCTGCGTTTGCCTTGGGGA

TTACCTTGCGTATAACCACGGATATGACAAAGCTAACACTAAATGGGAACAGGAGGTGCACAATGAGTACGTCAAG

AAAGCTGAGGCGACTGCGGATAAACAGGCTGCTATCAATGAAGTCTCACAGCAATACCAAGAAGACCTTGCGGCGCT

GGAGGGCAGCACTGATAGGGTCATTAATGATTTGCGTAACGATGGTAAGCGGCTGCGTGTCAAACTATCAGCCACCTC

ACGAGAACTCCAAGCTAACGGTGGATGCCTCGTTGATGGTCGAGCCGAACTTGACAAAGAGTTTAGTAGACGTCTTAT

CGGAGTAACTCAAAGAGGGGATGCGTGGATTAAGGCGCTCCAAAATACAATCAAAGAAATGCAACATAAGGAGAAA

CACAATGGCTAACGTAAGAATCAGAACCATATACACTCTCAACGGACGTGCACCAGATAGTCGTGACGTATGGGATA

AGAACAGTGTGGGCCTTCTAGTAGGTCGTATTGACCTTCCTGAGTCCTCTATGGTAACAGTAGAAGGATACACTAAGG

TAGAATATCAGGTGAAACTGTCTGATGGAGCTATGGAGGTGTACCCCAACTTCTCTGCATTCATCGCTGCGTGGAGCG

GAACCATTGAGTCTAAAGTTGGCAGTGGATATATGACTGTGTTCCTTAATAGAGCTAAAGGTGACACATTGGGTCTAG

TACAGATTGATGTACATAAAGAGTGGGATGACTCTACTCCAGACCCTGAGCCAGAACCGTCTCCTGAGATGGACGTAG

CGGGTATCACTAAGTTCTTTACGACCTATGTGGATAACCCTGAGTACCTCTTTGTGTGGAACGAGATGACCACTGTAG

GTGGTTCAGAACGTAACCGTCGTCTGCCGCACGAGATGTTTTGGTGTTGCTCTGGTTAACTCTATTAAGCATATGCGCCC

ACAGACCCTCAAGGCTCTTCAGGATGCGCTAGGTATCACAGCAACCACCGAGTAACATTAGGAGGCCTCAATGTCTAA

TCAAGCAAACAAGAATGCGCTCATAGTGGCGCAACTTAAGGGTGACTTCGTAGCTTTCTTGTTCGTCTTATGGAAGGC

TCTGAACTTACCTAAACCAACCAAGTGTCAGATTGATATGGCACGTACACTCGCTGATGGTGACCACAAGAAGTTTAT

CCTTCAGGCGTTCCGTGGTATCGGCAAGTCCTTTATCACTTGTGCGTTCGTTGTGTGGGTCTTATGGAGGGACCCTCAA

CTTAAGGTTCTCATTGTGTCTGCCTCTAAGGAACGTGCTGATGCTAACTCCATCTTCATCAAGAACATCATTGACCTGC

TACCGTTCCTCGCTGAGCTGAAACCTCGACCGGGACAGCGTGACTCTGTGATTAGCTTTGATGTTGGCTTAGCTAAACC

TGACCATTCTCCCTCTGTGAAGTCTGTAGGTATTACTGGTCAGTTGACTGGTAGCCGTGCTGACATTATCATTGCGGAT

GACGTTGAGGTGCCTGGTAACAGTTCTACAAGTTCTGCTCGTGAGAAGCTGTGGACGCTGGTAACTGAGTTTGCTGCA

TTACTTAAGCCATTACCAACCTCTCGTGTTATCTACCTAGGGACGCCTCAAACCGAGATGACTCTCTACAAGGAACTTG

AGGATAACAAAGGGTACAGCACAGTAATCTGGCCCGCACAGTATCCACGCAATGATGCTGAGGCTCTCTATTATGGG

GACCGCTTGGCTCCTATGCTTAAGGCTGAATATGATGAAGGCTTTGAGTTACTCCGTGGACAACCTACTGACCCTGTTC

GATTCGATACGGATGACCTTCGTGAACGTGAACTCGAATATGGCAAAGCTGGTTACACTCTTCAGTTCATGCTTAACC

CTAACCTTAGTGATGCCGAGAAGTACCCGTTACGTCTCCGTGACGCTATCGTGTGCGCTGTAGACCCTGAACGTGCCC

CATTGTCATACCAGTGGTTGCCAAACCGTCAGAACCGCAATGAGGAGCTTCCTAACGTGGGCCTGAAAGGTGATGACA

TCCATAGCTTCCATACGTGCTCCTCTAGAACCGCTGAGTACCAGTCTAAGATTCTTGTCATTGACCCCAGTGGTCGTGG

TAAGGATGAGACCGGGTATGCAGTCCTCTATTCCCTGAATGGCTACATCTACTTGATGAGGTCGGTGGGTTCCGTGG

AGGCTACGATGATGCTACCCTAGAGAAGCTCGCTAAGAAGGCCAAGCAGTGGAAGGTACAGACAGTAGTCCACGAGT

CCAACTTTGGGTGACGGTATGTTCGGTAAGATTTTCTCTCCTGTTCTCCTTAAGCACCACAAGGCTGCACTCGAAGAGAT

TCGAGCTAAGGGCATGAAGGAGATGCGAATCTGCGATACATTGAACCATTGATGGGCTCCCATAAGCTAATCATTCG

TGATGAAGTTATCCGTGAAGACTATCAGACTTCTCGTGACCTAGATGGGAAACACGATGTTCGCTACAGTGCGTTCTA

TCAGATGACCCGTATGACTCGTGAGCGAGGCCTGTAGCACACGATGACCGACTTGATGCTATCGCATTGGGTATCGA

ATGGCTACGCGAGGGGATGCTTGTAGACAGCAAGATAGGCGAAGAAGAGATGACCCTTGAGTTCCTTGAGGCACACA

TGGAGAAACAGACCATTGGTGGTGACCAGATTCATAGCCTTGATGTTGGTGGCGTGGACATCTATTATGAGGACGAGG

AAGGAGGCTCTAGCTTCATCGACTGGTAACAGCTACACAAACAGAAGGAGGTGTGAGTAATGAGAGTGGCAATTGAT

AGTATAACTGGTTGCCACATCTTCCTAGGGTGTAAGACTAAAGGTGGCTACGGTAGGATTCGTGTTAATGGCGTACAT

TGGATGGCCCATAGATACGCCCTGCACGTCCACCTGAAGCGTCCCTTAAAGGAAGGATGTGTGGTAACGCATTCGTGT

GATAACCCTTCGTGTGTGAACCCTGAGCACTTATCTGAGGGAACACAGAAAGAGAACATAGAGGACTGTAGGTCGAA

AGGTAGTATGGTCCATAGTGATTCTTCAGGTGGTTCTTCAGGTAAGCATCAAAGTCACATAGATAAGATTCAAAAGGC

ATTCAATAGACGACTGAAGGCTGTACTTGATGCTGATGGTACAGATTCTGAGGTTGCTAAGAGACTTGGCGTAAACAT

CCAGTGGGTCAAGAAGGCTCGGTCTGGTAGACTAAGTATGCACATAAAGTGACTTAATATTCACAATGGGCACCCTAT

GGAGTGAGGTGTAAAAACCTAATGAAATCAAGGGTGTCCCTAATACGACTCACTATTGGGAAGGGCCCCCTAAGACA

ACTTAAAGACACTTAAAGATTGCATATGCATAGTGCATACTTAGTGACTACTCTTAGTGACTACTCTTAGTGACTACTC

TTAGTGACTACTCTTAGTGACTACTCTTAGTGACTACTCTTAGTGACTACTCTTAGTGACTACTCTTAGTGACTACTCTT

AGTGACTACTCTTAGTGACTACTCTTAGAGACCCTTACAGTGAGGGATGATAGTAATAGTAATTACATCCCCACTATC

AGTGTAACCATCAAGTATAACAAGGAGGACCGCTCTATGCGCTTACTATACGTACTCAAGACACTGGCAACGCATCGAA

CGACTTATAAATTTCTCGTTGTACTTGCTGGTTCCATTGGTATCACATCTGGTTCTGAACACATCGGTGAACTGGAAAC

TCTGCTTTGTTCTATACTCACTTGTGTTCATTAGACTACTCATTGACGCTTAGTGTGCTCGTAGTGATTATGGTCGATAT

ACATCTTCCCCTATAATCACTGTAAGAAGTCAACTCTAAAGAGTAACCCTTATGTGCCCTCTATGAGCCTACAGGGTCTC

TTAGAGTCTATCAAGGTCTCTTAGTACCTCTTAGTACCTCTTAGTGTCTCTATGGGCCTCTAGGGTGCCCTCAGGACCA

CCTTATGATAGACACTCCAAGTGGTTAACCTTATGAGACTCTTGGTGTGCACCCTTTAAGAATCCTACAGAAAAATCTG

AGTGAGTATCTAATAGTACATACAAAGTGTTTCCCCCCTATGGCCTCCTTATGCTCACCCCAAAGAGAGCACCCCTATG

GTGACTTTAGGTTTACTTTAGGTGTACTTGAGGTGTACTTTAGGTGTACTTAATGCCTAACCTTATGTGTGACATTGGG

TTTACTACTAGATGTAGTGCTTAGGGGGGACTTTAGGGGACTATATGTAGGCCTCTTTCTGTCTCTGACTGTA

SEQ ID No.2: Sequence between nucleotides 19,197 and 19,933 of SEQ ID No.1

ATGTCCGGTGAATCTAATGCTG ATGTTTATGCATCTTTCGGTGTGAACTCTGCGGTTGTCGGTGGGTCTACTCCTACTG

ACCACGAACAGGCTATGCTTGAACTCAATGTTGCTGCCCGTGATGGCGATGATGCAATCGAGATAGTAGGCAATGATG

ACCCTTATGGTAATCCAGACCCGTTT GGTGAAGAAGAAGACGAGAAT CGTACTGAGATTCGTATCTCCACTGATGGTG

AACCTGAAGAGACTGAAGAGGGTGAGATTGATTACTCTGAGAGCACTGAAGGAGGCTCAGAAGAGTTTTGCACCTGTA

GGTGATGTTCCAGAAGAACTAAGTGCAGCCTCAAGTCAACTCGAAGAGCATGAGACGGGCTTCGAAGAGATGGTCAA

CCAGGCTGCTGAACGTGGTCTGTCTGAAGAAACTATTGTCCGTATCCAACAGGAATATGGTGAAGAGGGTCTGTCCAA

GGAATCCTATGAAGAACTGGCTAAGGCTGGTTACTCTAAGTCCTTCGTTGACTCCTACATTCGTGGACAGGAAGCACT

GGTCGAACAGTACGTTCAGTCTGTTATGGAGTATGCTGGTGGTGAGTCACAGTTCCAGGCAATCTATAGCCACCTCGA

AGTGTCCAACCCGGATGCTGCCGAGTCTCTGGTTAATGCTCTGGAAAACCGT GACCTAGCGACCGTTAA GGCTATCGT

GAATCTTGCTGGG GCAAGCCGTACTAAGACTTTTGG

SEQ ID No.3: Sequence between nucleotides 19,380 and 19,889 of SEQ ID No.1

GGTGAAGAAGAAGACGAGAAT CGTACTGAGATTCGTATCTCCACTGATGGTGAACCTGAAGAGACTGAAGAGGGTGA

GATTGATTACTCTGAGAGCACTGAAGGAGGCTCAGAAGAGTTTCACCTGTAGGTGATGTTCCAGAAGAACTAAGTGC

AGCCTCAAGTCAACTCGAAGAGCATGAGACGGGCTTCGAAGAGATGGTCAACCAGGCTGCTGAACGTGGTCTGTCTG

AAGAAACTATTGTCCGTATCCAACAGGAATATGGTGAAGAGGGTCTGTCCAAGGAATCCTATGAAGAACTGGCTAAG

GCTGGTTACTCTAAGTCCTTCGTTGACTCCTACATTCGTGGACAGGAAGCACTGGTCGAACAGTACGTTCAGTCTGTTA

TGGAGTATGCTGGTGGTGAGTCACAGTTCCAGGCAATCTATAGCCACCTCGAAGTGTCCAACCCGGATGCTGCCGAGT

CTCTGGTTAATGCTCTGGAAAACCGT GACCTAGCGACCGTTAAG

SEQ ID No.4: Sequence between nucleotides 19,197 and 19,379 of SEQ ID No.1

ATGTCCGGTGAATCTAATGCTG ATGTTTATGCATCTTTCGGTGTGAACTCTGCGGTTGTCGGTGGGTCTACTCCTACTG

ACCACGAACAGGCTATGCTTGAACTCAATGTTGCTGCCCGTGATGGCGATGATGCAATCGAGATAGTAGGCAATGATG

ACCCTTATGGTAATCCAGACCCGTTT

SEQ ID No.5: Sequence between nucleotides 19,890 and 19,933 of SEQ ID No.1

GCTATCGTGAATCTTGCTGGG GCAAGCCGTACTAAGACTTTTGG

Claims (18)

1. A pair of nucleic acid primers for identifying Yersinia pestis-specific phages in biological specimens, wherein the target nucleic acid sequence amplified by the primers is between the 19,197th and 19,933rd nucleotides of SEQ ID No. 1 (SEQ ID No. 2) or any fragment thereof, wherein the length of the fragment is not less than 100 bp. 2. The nucleic acid primer according to claim 1, wherein the length of the target nucleic acid sequence amplified by the primer or any fragment thereof is 100bp to 737bp, preferably the length is 200bp to 737bp, 300bp to 737bp, 400bp to 737bp, 500bp to 737bp, 600bp to 737bp, or 700bp to 737bp, for example, a length of 100, 150, 200, 250, 300, 350, 400, 450, 500, 510, 550, 600, 650, 700bp or 737bp. 3. The nucleic acid primer according to claim 1, wherein the target nucleic acid sequence amplified by the primer is the sequence between the 19,380th and 19,889th nucleotides of SEQ ID No. 1 (SEQ ID No. 3) or any fragment thereof, wherein the length of said fragment is not less than 100 bp. 4. The nucleic acid primer according to claim 3, wherein the length of the target nucleic acid sequence amplified by the primer or any fragment thereof is 100bp to 510bp, preferably the length is 200bp to 510bp, 300bp to 510bp, 400bp to 510bp, or 500bp. to 510bp, e.g. 100, 150, 200, 250, 300, 350, 400, 450, 500 or 510bp in length. 5. The nucleic acid primer according to claim 1, wherein in the pair of primers, the forward primer corresponds to the sequence between the 19,197th to 19,379th nucleotide of SEQ ID No. 1 (SEQ ID No. .4) or any fragment thereof, and the reverse primer corresponds to the complementary sequence of the sequence between nucleotides 19,890 to 19,933 of SEQ ID No. 1 (SEQ ID No. 5) or any fragment thereof. 6. The nucleic acid primer according to any one of claims 1-5, wherein the length of the primer is each independently 10bp to 50bp, such as 15bp to 50bp, such as 15bp to 40bp, 15bp to 35bp, 15bp to 30bp, 15bp to 25bp, or 18bp to 23bp, such as 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30bp. 7. The nucleic acid primer according to any one of the above claims, wherein the primer is DNA. 8. The nucleic acid primer according to any one of the above claims, wherein the primer sequence is selected from: F da:5’-ATgTCCggTgAATCTAATgCTg-3’ R da: 5’-CCAAAAgTCTTAgTACggCTTgC-3’ where F and R represent forward and reverse primers respectively; or F xiao：5’-ggTgAAgAAgAAgACgAgAAT-3’ R xiao：5’-CTTAACggTCgCTAggTC-3’ where F and R represent forward and reverse primers respectively. 9. A combination of two pairs of nucleic acid primers used to identify Yersinia pestis-specific phages in biological specimens, including: (1) A first pair of primers, wherein the forward primer corresponds to the sequence between 19,197th and 19,379th nucleotide of SEQ ID No. 1 (SEQ ID No. 4) or any fragment thereof, and the reverse primer A complementary sequence corresponding to the sequence between 19,890th and 19,933rd nucleotides of SEQ ID No. 1 (SEQ ID No. 5) or any fragment thereof, wherein the forward primer and the reverse primer have respective lengths Independently 10bp to 50bp, such as 15bp to 50bp, such as 15bp to 40bp, 15bp to 35bp, 15bp to 30bp, 15bp to 25bp, or 18bp to 23bp, such as 15, 16, 17, 18, 19, 20, 21, 22 , 23, 24, 25, 26, 27, 28, 29 or 30bp; and (2) A second pair of primers, wherein the target nucleic acid sequence amplified by the primers is the sequence between the 19,380th and 19,889th nucleotides of SEQ ID No. 1 (SEQ ID No. 3) or any fragment thereof , wherein the length of the fragment is not less than 100bp, and the length of the primers is each independently 10bp to 50bp, such as 15bp to 50bp, such as 15bp to 40bp, 15bp to 35bp, 15bp to 30bp, 15bp to 25bp, or 18bp to 23bp, such as 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30bp. 10. The combination of two pairs of nucleic acid primers according to claim 9, comprising: (1) The first pair of primers, which is F da: 5’-ATgTCCggTgAATCTAATgCTg-3’ R da: 5’-CCAAAAgTCTTAgTACggCTTgC-3’ where F and R represent forward and reverse primers respectively; and (2) The second pair of primers, which is F xiao: 5’-ggTgAAgAAgAAgACgAgAAT-3’ R xiao：5’-CTTAACggTCgCTAggTC-3’ where F and R represent forward and reverse primers respectively. 11. A detection kit for identifying Yersinia pestis-specific phages in biological specimens, which contains the nucleic acid primer or primer combination according to any one of claims 1-10. 12. The detection kit according to claim 11, which contains the nucleic acid primer or primer combination according to any one of claims 1-10 and instructions for use. 13. The detection kit according to claim 11, which contains the nucleic acid primer or primer combination according to any one of claims 1-10, instructions for use and/or reagents for PCR reaction, such as polymerase, purification Water, buffer and dNTP mixture, and optional positive control. 14. The detection kit according to any one of claims 11-13, wherein the kit can be in the form of a single package or a virtual package. 15. A method for identifying Yersinia pestis-specific phages in biological specimens, the method comprising the following steps: a) grind tissue samples and perform nucleic acid extraction; and b) Use the primer or primer combination according to any one of claims 1 to 10 or the detection kit according to any one of claims 11 to 14 to perform PCR detection on the nucleic acid sample extracted in step a). 16. The method of claim 15, further comprising the steps of: c) Proliferate and culture the sample that is positive in the primer test in step b) in Yersinia pestis; d) Separate the phage proliferation fluid; and e) Phage lysis experiments in Y. pestis. 17. The method of claim 16, wherein steps c) to e) comprise the following steps: (1) Add the tissue filtrate that was positive in the primer test in step b) to the freshly prepared Yersinia pestis bacterial solution, (2) Shake the above mixture to mix it evenly, (3) The resulting mixture is cultured statically at 28°C. (4) Pass the cultured phage proliferation solution through a 0.45μm filter, (5) Pass the obtained filtrate through a 0.22μm filter, (6) Add the resulting filtrate to a freshly prepared Y. pestis-inoculated Buchner plate, and (7) Observe the phage lysis to determine whether it is a Yersinia pestis-specific phage. 18. Use of the nucleic acid primer or primer combination according to any one of claims 1 to 10 in the preparation of a detection kit for identifying Yersinia pestis-specific phages in biological specimens.