WO2013071520A1 - Method and system of virus detection - Google Patents

Abstract

Provided are a method and a system of virus detection. The method for virus detection comprises the following steps: for a sample containing a viral nucleic acid, preparing a DNA sequencing library; using the Ion Torrent technology to perform DNA sequencing on the DNA sequencing library to obtain sequence information of the viral nucleic acid; and determining the type of the virus according to the sequence information of the viral nucleic acid.

Description

 Method and system for virus detection

 The invention relates to the field of molecular biology. In particular, the present invention relates to methods and systems for virus detection.

Background technique

 Cervical cancer is the second highest incidence of cancer in women worldwide, and its incidence is mainly related to the persistent infection of human papillomavirus (HPV). There are approximately 500,000 new cases worldwide and 250,000 deaths each year, of which 2/3 are in developing countries. More than 100 HPV types have been characterized to infect the skin (skin type) or the mucous membranes of the respiratory and anal genital tract (mucosal type) ), more than 40 HPV types can infect the cervix. Based on induced benign, premalignant or malignant lesions, HPV is classified into low-risk (eg, HPV6, 11, 42, 43, and 44) and high-risk (eg, types 16, 18, 31, 33, and 45). Therefore, early detection and correct typing of HPV infection is essential for the prevention of cervical cancer.

 However, the current HPV detection method still needs to be improved.

Summary of the invention

 The present invention is directed to solving at least one of the problems of the prior art. To this end, in one aspect of the invention, a method for efficiently detecting a virus is proposed. The method comprises the steps of: preparing a DNA sequencing library for a sample comprising a viral nucleic acid; performing DNA sequencing on the DNA sequencing library by Ion Torrent technology to obtain sequence information of the viral nucleic acid; and sequence information based on the viral nucleic acid Determine the type of the virus. With this method, the Ion Torrent sequencing technology can be used to quickly and accurately detect the virus, and the cost is low, the operation is simple, and it is easy to promote. The term "detection" as used herein shall be understood broadly and may be either a diagnostic test or a non-diagnostic test. The meaning of diagnostic testing means that the test results can be directly used as a basis for whether or not the disease is present.

 According to still another aspect of the present invention, the present invention also provides a system for virus detection, comprising: a DNA sequencing library preparation device for preparing a DNA sequencing library against a sample containing a viral nucleic acid; a sequencing device, coupled to the DNA sequencing library preparation device, for sequencing the DNA sequencing library to obtain sequence information of the viral nucleic acid; and an analysis device, the analysis device and the sequencing The device is ligated for determining the type of the virus based on sequence information of the viral nucleic acid, wherein the sequencing device is a device suitable for implementing the Ion Torrent sequencing technology. With this system, the virus detecting method of the embodiment of the present invention can be effectively carried out, thereby effectively detecting the virus in the sample and detecting the type of virus contained therein.

In addition, according to the present invention, the present invention also proposes a DNA tag (herein, simply referred to as "tag") for constructing a DNA sequencing library. According to an embodiment of the invention, the invention proposes a set of isolated DNA tags. These isolated DNA tags are composed of the nucleotides shown in SEQ ID NOS: 1-56, respectively. In the present specification, these DNA tags are respectively named DNA IndexN, wherein any integer of N = 1-56, the sequence of which is shown in Table 1 below. Using the DNA tag described above in accordance with an embodiment of the present invention, the source of the sample of DNA can be accurately characterized by linking the DNA tag to the sample DNA or its equivalent. Thus, by using the above DNA tag, a DNA sequencing library of a plurality of samples (herein, sometimes referred to as a "sequencing library") can be simultaneously constructed, thereby enabling simultaneous sequencing based on DNA sequencing libraries derived from different samples. The DNA tag classifies the DNA sequence of the DNA sequencing library to obtain DNA sequence information of various samples, thereby making full use of high-throughput sequencing technology, such as using Ion Torrent sequencing technology, and simultaneously performing multiple DNA sequencing libraries. Sequencing increases the sequencing efficiency and throughput of DNA sequencing libraries. The inventors have surprisingly found that the construction of a DNA sequencing library using a DNA tag according to an embodiment of the present invention enables precise discrimination of a plurality of DNA sequencing libraries, and the resulting sequencing data results are very stable and reproducible.

 DNA tag (DNA IndexN ) sequence (5, → 3, direction)

 Name Name Sequence (SEQ ID NO: ) Sequence (SEQ ID NO: )

 DNA Indexl DNA Index29 TCATCGTAGA(29)

 GCTGCGACTC(l)

 DNA Index2 DNA Index30 TAGCATCTGT(30)

 GTGTAGATAC(2)

 DNA Index3 DNA Index31 TAGTAGTCGT(31)

 CTGATATCTA(3)

 DNA Index4 DNA Index32 CTATACGTGC(32)

 ACGATGCTAT(4)

 DNA Index5 DNA Index33 CGACTGTAGA(33)

 TAGACTAGAC(5)

 DNA Index6 DNA Index34 GATGTCATGT(34)

 CTGTCTGTGT (6)

 DNA Index7 DNA Index35 GTCTCGACTG(35)

 GCATACTGAC(7)

 DNA Index8 DNA Index36 AGCTGACGAT(36)

 CTGCTCGCAT(8)

 DNA Index9 DNA Index37 ATGATATAGT(37)

 CATGAGTAGA(9)

 DNA IndexlO DNA Index38 ATGTGCTCTA(38)

 TCTCACTATG(IO)

 DNA Indexl 1 DNA Index39 CTCACTCGAT(39)

 TGTACTACTA(ll)

 DNA Indexl2 DNA Index40 GCTGCGACTC(40)

 GTAGACTAGT(12)

 DNA Indexl3 DNA Index41 GAGTCATGTC(41)

 ATATGCTACT(13)

 DNA Indexl4 DNA Index42 CATACGCTCA(42)

 CACTCGCTGT(14)

 DNA Indexl5 DNA Index43 CACTCTCGTC(43)

 CATCACGCAC(15)

 DNA Indexl6 DNA Index44 GCACTAGATG(44)

 AGCATGTGAT(16)

 DNA Indexl7 DNA Index45 AGTACGCATG(45)

AGCTAGTAGA(17) DNA Indexl8 DNA Index46 TCTGTGACGT(46)

 GCTATGTAGT(18)

 DNA Indexl9 DNA Index47 TAGCTCATCT(47)

 TACGATGATG (19)

 DNA Index20 DNA Index48 AGCATACACT(48)

 TACGCTGTAC(20)

 DNA Index21 DNA Index49 GCTATAGTCA(49)

 TATGTGTACT(21)

 DNA Index22 DNA Index50 CGTCTCATGC(50)

 TGACTCAGAC(22)

 DNA Index23 DNA Index51 GCTACTACGT(51)

 TCGTAGCTCA(23)

 DNA Index24 DNA Index52 GAGTGTACTA(52)

 GAGACTCGTA(24)

 DNA Index25 DNA Index53 GTCATACGTG(53)

 CTAGATGTCA(25)

 DNA Index26 DNA Index54 TATGAGAGAT(54)

 GATGACTCTC(26)

 DNA Index27 DNA Index55 ATCTGAGTAC(55)

 TCAGTCGCAC(27)

 DNA Index28 DNA Index56 CGATAGCATC(56)

TGTAGTGAGT (28) According to another aspect of the invention, the invention provides a set of isolated PCR forward label primers for introducing the above DNA tag into sample DNA or an equivalent thereof. Each of the set of isolated PCR forward tag primers according to an embodiment of the invention independently comprises: a first nucleic acid sequence, the first nucleic acid sequence being one selected from the group consisting of SEQ ID NOs: 57-62 And a second nucleic acid sequence, the second nucleic acid sequence being one selected from the group consisting of an isolated DNA tag according to an embodiment of the present invention, wherein the second nucleic acid sequence is 5 of the first nucleic acid sequence The ends are connected. In the present specification, each of the set of PCR forward tag primers comprises a DNA tag according to an embodiment of the present invention as described above, and PCR can be performed by PCR reaction using a PCR forward tag primer. The primer is introduced into the DNA of the sample or its equivalent, thereby introducing the corresponding DNA tag into the DNA or its equivalent. The first nucleic acid sequence constituting the PCR forward label primer is shown in Table 2 below. According to yet another aspect of the invention, the invention also provides a set of isolated PCR reverse tag primers for introducing the above DNA tag into sample DNA or an equivalent thereof. Each of the set of isolated PCR reverse tag primers according to an embodiment of the invention independently comprises: a third nucleic acid sequence, said third nucleic acid sequence being one selected from the group consisting of SEQ ID NOs: 63-67; a fourth nucleic acid sequence, the fourth nucleic acid sequence being one selected from the group consisting of an isolated DNA tag according to an embodiment of the invention, wherein the fourth nucleic acid sequence is linked to the 3' end of the third nucleic acid sequence . In the present specification, each of the set of PCR reverse-label primers has a DNA tag according to an embodiment of the present invention as described above, and PCR can be reverse-labeled by PCR using a PCR reverse-label primer. The primer is introduced into the DNA of the sample or its equivalent, thereby introducing the corresponding DNA tag into the DNA or its equivalent. The third nucleic acid sequence constituting the PCR reverse tag primer is shown in Table 2 below. Table 2 First nucleic acid sequence and third nucleic acid sequence (5, → 3, direction)

 Using the above-described set of isolated PCR forward tag I and a set of isolated PCR reverse tag primers according to an embodiment of the present invention, can effectively introduce a DNA tag into the DNA of the sample or its equivalent, This enables the construction of a DNA sequencing library with a DNA tag. In addition, the inventors have surprisingly found that when constructing DNA sequencing libraries containing various DNA tags using PCR forward tag primers with different tags for the same sample, the resulting sequencing data results are stable and repeatable. Very good; Similarly, when constructing DNA sequencing libraries containing various DNA tags using PCR reverse tag primers with different tags, the resulting sequencing data results are also very stable and reproducible. According to an embodiment of the present invention, when PCR amplification reaction is carried out by using at least one of PCR forward label primer and PCR reverse label primer, in order to construct a DNA sequencing library of sample DNA, PCR can be used to positively label primers and The PCR reverse-label primer introduces a DNA tag into a DNA sequencing library of the sample DNA, thereby enabling simultaneous construction of a DNA sequencing library of a plurality of sample DNAs. Thus by using a PCR forward tag with a DNA tag according to an embodiment of the invention? PCR reaction can be performed on at least one of the PCR and reverse PCR primers, and a DNA sequencing library of a plurality of samples can be constructed, so that the DNA sequencing library can be distinguished according to the different labels in different DNA sequencing libraries, and finally a large number of samples can be realized. Hybrid sequencing to meet the needs of high-throughput sequencing, thereby reducing sequencing costs.

The additional aspects and advantages of the invention will be set forth in part in the description which follows. DRAWINGS The above and/or additional aspects and advantages of the present invention will become apparent and readily understood from

 Figure 1: A flow diagram showing a method of constructing a DNA sequencing library in accordance with one embodiment of the present invention. Figure 2: shows an electropherogram of a partially PCR amplified product during the construction of a DNA sequencing library in accordance with one embodiment of the present invention.

 Figure 3: shows a schematic diagram of a template structure applied to Ion Torrent sequencing in accordance with one embodiment of the present invention. Figure 4: A schematic diagram showing a system for genotyping HPV in accordance with one embodiment of the present invention. Detailed description of the invention

 The embodiments of the present invention are described in detail below, and the examples of the embodiments are illustrated in the drawings, wherein the same or similar reference numerals are used to refer to the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the drawings are intended to be illustrative of the invention and are not to be construed as limiting.

 It should be noted that the terms "first" and "second" are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or implicitly indicating the number of technical features indicated. Thus, features defining "first" and "second" may explicitly or implicitly include one or more of the features. Further, in the description of the present invention, the meaning of "plurality" is two or more unless otherwise stated.

 Virus detection method

 According to an embodiment of the present invention, the present invention provides a virus detecting method, comprising:

 First, a DNA sequencing library is prepared for a sample containing viral nucleic acid.

 Next, the DNA sequencing library constructed is subjected to DNA sequencing using Ion Torrent technology to obtain sequence information of the viral nucleic acid.

 Finally, the type of the virus is determined based on the sequence information of the viral nucleic acid. According to an embodiment of the present invention, after obtaining the sequence information of the viral nucleic acid, the method of determining the virus type is not particularly limited, and the viral nucleic acid sequence can be aligned with a known viral nucleic acid database, such as the genome database of HPV.

The term "viral nucleic acid" as used herein, shall be taken broadly and may refer to a nucleic acid extracted directly from a virus, such as a whole genome of a virus or a portion thereof, or may be extracted from a host cell infected with a virus. A whole genome or portion thereof of a host cell containing viral nucleic acid information. Further, the type of the viral nucleic acid is not particularly limited and may be DNA or RNA. According to an embodiment of the invention, RNA can be converted to a DNA sequence by conventional reverse transcription techniques. According to an embodiment of the invention, the virus is human papillomavirus (HPV). According to a specific example, the sample comprising viral nucleic acid is a whole genetic DNA sample of a female biological sample. Preferably, the woman The sex biological sample is at least one selected from the group consisting of female cervical exfoliated cells and cervical cancer tissues. The inventors have surprisingly found that the HPV nucleic acid information contained in the host cell can be efficiently detected using the method of the present invention. One skilled in the art can extract whole genomic DNA from a biological sample by any known method. According to a specific example of the invention, whole genome DNA samples were performed using a KingFisher automatic extractor.

 According to an embodiment of the present invention, a method which can be used for constructing a DNA sequencing library is not particularly limited as long as the constructed library can be applied to Ion Torrent sequencing technology. Preferably, the preparation is carried out by a method comprising the following steps (refer to FIG. 1): the sample containing the viral nucleic acid is subjected to a PCR amplification reaction to obtain an amplification product, wherein the PCR amplification reaction uses a forward primer And a reverse primer, the forward primer and the reverse primer are specific for the virus; the amplification product is end-repaired to obtain an end-repaired amplification product; The amplification product is ligated to a sequencing adaptor to obtain a ligation product having a sequencing adaptor; and the ligation product having the sequencing adaptor is isolated and recovered, the ligation product having the sequencing linker constituting the DNA sequencing library. By using virus-specific PCR forward primers and reverse primers, sequences of specific regions of the virus can be obtained, thereby improving the efficiency of virus detection. One skilled in the art can determine the corresponding primer sequence ij ij based on the sequence of the viral nucleic acid. According to an embodiment of the invention, it is directed to an HPV virus. The forward primer which can be used includes a first nucleic acid sequence which is one selected from the group consisting of SEQ ID NOs: 57-62. The reverse primer which can be used includes a third nucleic acid sequence which is one selected from the group consisting of SEQ ID NOS: 63-67. The inventors have surprisingly found that by using the above primers, it is possible to significantly and efficiently amplify the HPV viral nucleic acid sequence in female biological tissues for detection and typing. In addition, according to an embodiment of the present invention, it is also possible to introduce a DNA tag into a sequencing library by a PCR reaction, thereby enabling virus detection of a plurality of samples at the same time. To this end, according to an embodiment of the present invention, the forward primer may further include a second nucleic acid sequence which is one selected from the group consisting of SEQ ID NOs: 1-56, wherein the second nucleic acid sequence and the first nucleic acid The 5th end of the sequence is connected. According to an embodiment of the present invention, the reverse primer may further comprise a fourth nucleic acid sequence, wherein the fourth nucleic acid sequence is one selected from the group consisting of SEQ ID NOs: 1-56, wherein the fourth nucleic acid sequence and the The three nucleic acid sequences are ligated to the ends. The DNA label will be described in detail later.

According to an embodiment of the present invention, before the end product is subjected to end repair, a step of purifying the amplified product, for example, may be carried out by using Qiagen DNA Purification Kit according to the manufacturer's instructions. Thereby, the efficiency of end-repairing the amplified product can be improved, thereby further improving the efficiency of constructing the sequencing library. One skilled in the art can select a commercially available kit for end repair of the amplified product. According to some embodiments of the invention, the step of purifying the end-repaired amplification product is further included prior to linking the end-repaired amplification product to the sequencing linker. According to a specific example of the invention, the ligation product with the sequencing linker is about 180-200 bp in length. Thereby, the efficiency of constructing the sequencing library can be further improved. In addition, this application The inventors have surprisingly found that when a ligation product of about 180-220 bp in length is used, the efficiency of subsequent sequencing can be significantly improved, in particular, the efficiency of sequencing using Ion Torrent. According to a specific example of the present invention, the ligation product having the sequencing linker was separated and recovered by electrophoresis at 100 V for 2 hours using a 2.0% agarose gel. According to an embodiment of the present invention, the type of the sequencing linker is not particularly limited, and according to a specific embodiment, a sequencing linker having the following double-stranded nucleotide sequence can be used:

 The P5 linker has a double-stranded structure, which are:

5 ' -CCACTACGCCTCCGCTTTCCTCTCTATGGGCAGTCGGTGAT ( SEQ ID NO: 68 ) 3 ' -T*T*GGTGATGCGGAGGCGAAAGG AGAGATACCCGTC AGCC ACTA ( SEQ ID NO: 69 )

 The A linker sequence has a double-stranded structure, which are:

 5 ' -TTCCATCTCATCCCTGCGTGTCTCCGACTC AG ( SEQ ID NO: 70 )

 3 '-T*T* AAGGTAGAGTAGGG ACGC ACAGAGGCTGAGTC (SEQ ID NO: 71). The T with an asterisk in SEQ ID NOS: 69 and 71 indicates that the T base does not have a phosphate group. Thus, according to an embodiment of the present invention, after obtaining a ligation product having a sequencing linker, the obtained ligation product having the sequencing adaptor can be subjected to a nick translation reaction, which can further improve the efficiency of constructing the library, and the efficiency of subsequent sequencing. One of skill in the art can select any commercially available kit for nick translation, such as Platinum® PCR SuperMix High Fidelity, from the Ion XpressTM Fragment Library Kit.

According to an embodiment of the present invention, sequencing the DNA sequencing library of the DNA sample further comprises the step of preparing an Ion Torrent sequencing template by an emulsion PCR reaction. Preferably, the step of diluting the DNA sequencing library is further included prior to performing the emulsion PCR amplification. More preferably, in the emulsion PCR amplification reaction system, the concentration of the ligation product having the sequencing linker is 560*10 ⁶ molecules/reaction system. The inventors have found that when amplification is carried out by conventional PCR, the amplification efficiency of DNA molecules of different lengths sometimes differs markedly in the same reaction system.

 To this end, the inventors developed an emulsion PCR reaction that can be applied to Ion Torrent sequencing technology. According to an embodiment of the invention, the Ion Torrent sequencing template is prepared by emulsion PCR reaction, further comprising the following steps:

First, a water-in-oil emulsion is formed, the water-in-oil emulsion comprising a plurality of discontinuous aqueous compartments, at least a portion of the plurality of discontinuous aqueous compartments comprising: magnetic particles, the DNA sequencing library A portion of an oligonucleotide primer for amplifying the DNA sequencing library, wherein at least a portion of the oligonucleotide primer is linked to the magnetic particle. The method for forming a water-in-oil emulsion may be to form a water-in-oil emulsion by high-speed shaking of an aqueous solution containing an magnetic particle, a sequencing library, an oligonucleotide primer, and a reagent required for performing a PCR reaction, and an oil phase. The aqueous compartment of each water-in-oil emulsion can be used as a PCR reaction vessel, whereby a plurality of discrete aqueous compartments can independently undergo PCR amplification reactions, which can effectively perform each DNA molecule of the sequencing library. Efficient amplification, and due to the attachment of primers on the magnetic particles, multiple clones of DNA molecules can be formed on the magnetic particles, and after the magnetic particles are recovered, single-stranded DNA can be obtained by cleavage of the double-stranded DNA into single-stranded DNA. Magnetic particles, which in turn can be effectively applied to Ion Torrent sequencing technology. According to one embodiment of the invention, each of the plurality of discrete aqueous compartments contains at most one DNA molecule of the DNA sequencing library. Thereby, it can be achieved that the clones of the plurality of DNA molecules carried on the same magnetic particle are all derived from the same DNA molecule, thereby further improving the efficiency and accuracy of sequencing. According to an embodiment of the present invention, the type of magnetic particles to be used is not particularly limited, and according to a specific example of the present invention, Ion SphereTM particles can be used. In addition, according to an embodiment of the present invention, it may be determined according to the sequence of the DNA sequencing library, for example, the oligonucleotide for amplifying the DNA sequencing library may be determined by the sequence of the sequencing linker at both ends of the DNA molecule of the DNA sequencing library. The sequence of the primers. According to a specific example of the present invention, an oligonucleotide primer for amplifying the DNA sequencing library includes a first oligonucleotide primer and a second oligonucleotide primer, wherein the first oligonucleotide primer The sequence is TTCCATCTCATCCCTGCGTGTCTCCGACTCAG (SEQ ID NO: 72), and the first oligonucleotide primer is linked to the magnetic particle; the sequence of the second oligonucleotide primer is CCTATCCCCTGTGTGCCTTGGCAGTCTCAG-CCTCTCTATGGGCAGTCGGTGAT (SEQ ID NO: 73) And the second oligonucleotide primer is in a free state in the aqueous compartment. Thus, according to an embodiment of the present invention, for HPV, magnetic particles carrying single-stranded DNA for Ion Torrent sequencing can be efficiently prepared as an Ion Torrent sequencing template, thereby further improving the efficiency of detecting HPV virus.

 Next, the water-in-oil emulsion is placed under thermal cycling conditions suitable for amplifying the DNA sequencing library to form magnetic particles on which the DNA sequencing library is carried. According to an embodiment of the present invention, emulsion PCR is carried out to amplify the PCR conditions of the DNA sequencing library, i.e., the thermal cycle conditions are not particularly limited. According to a particular embodiment of the invention, it comprises the following:

 Step 1: 94 °C 6min

 Step 2: 94 °C 30sec

 Step 3: 58 °C 30sec

 Step 4: 72 °C 90sec

 Repeat steps 2-4, 40 cycles; and

 Step 5: 94 °C 30sec

 Step 6: 68 °C 6min

Repeat steps 5 and 6, 5 cycles. The inventors have surprisingly found that this thermal cycling condition is particularly suitable for the detection of HPV.

 Then, the magnetic particles carrying the DNA sequencing library thereon are placed under conditions suitable for double-stranded DNA cleavage, for example, the water-in-oil emulsion can be contacted with a lysate containing NaOH and Tween An aqueous solution of -20, the final concentrations of the NaOH and Tween-20 are 125 mM and 0.1% by weight, respectively, in order to obtain magnetic particles carrying single-stranded DNA thereon, and the magnetic particles carrying the single-stranded DNA thereon constitute an Ion Torrent sequencing template .

 After obtaining the magnetic particles carrying the single-stranded DNA thereon, according to an embodiment of the present invention, the steps of carrying the magnetic particles carrying the single-stranded DNA thereon may be further included, thereby further improving the efficiency of subsequent sequencing using the Ion Torrent sequencing technique. . Those skilled in the art can perform enrichment of the magnetic particles by any method depending on the nature of the magnetic particles selected. According to an embodiment of the present invention, magnetic particles carrying single-stranded DNA thereon can be enriched with MyOneTM beads. The inventors have found that using MyOneTM beads can effectively enrich Ion SphereTM particles carrying single-stranded DNA and can effectively improve the efficiency of HPV detection.

 DNA label

 According to one aspect of the present application, the present invention proposes a number of isolated DNA tags. According to an embodiment of the present invention, these isolated DNA tags are each composed of the nucleotide sequence shown in SEQ ID NOS: 1-56. In the present specification, these DNA tags are respectively named DNA IndexN, wherein any integer of N=l-56, the sequence of which is shown in Table 1 above, and will not be described herein.

 The term "DNA" as used in the present invention may be any polymer comprising deoxyribonucleotides including, but not limited to, modified or unmodified DNA. Using a DNA tag according to an embodiment of the present invention, a DNA sequencing library having a tag is obtained by linking the DNA tag to the DNA of the sample or its equivalent, and the DNA sequence of the sample DNA and the tag can be obtained by sequencing the DNA sequencing library. The sequence, and thus the sequence based on the tag, can accurately characterize the source of the sample of DNA. Thus, by using the above DNA tag, a DNA sequencing library of a plurality of sample DNAs can be simultaneously constructed, and the DNA sequence of the sample DNA can be synthesized based on the DNA tag by mixing and sequencing the DNA sequencing library derived from different sample DNAs. Classification, obtaining sequence information of DNA of a plurality of sample DNAs. This allows for the full use of high-throughput sequencing technologies, such as the use of Ion Torrent sequencing technology to simultaneously sequence multiple sample DNAs, thereby increasing the efficiency and throughput of high-throughput sequencing technologies and reducing the sequence information for determining sample DNA. the cost of. The expression "DNA tag is linked to the DNA of the sample or its equivalent" as used herein should be understood in a broad sense. It includes a DNA tag that can be directly linked to the DNA of the sample to construct a DNA sequencing library, and can also have DNA with the sample. A nucleic acid of the same sequence (for example, may be the corresponding RNA sequence or cDNA sequence, which has the same sequence as the DNA).

The inventors of the present application conducted a large number of screening work and selected an embodiment according to the present invention. A set of isolated DNA tags suitable for use in the Ion Torrent sequencing technique, which consist of the nucleotide sequences set forth in SEQ ID NOs: 1-56, respectively. The sequence is as shown in Table 1 above, and will not be described again. In general, different labels need to be developed for different sequencing technologies to be able to be used to simultaneously sequence multiple samples. There are currently no reports on the construction of DNA sequencing libraries for the application of these tags to sample DNA and sequencing by Ion Torrent. Therefore, the present invention also proposes the use of the above DNA tag for constructing an Ion Torrent sequencing library.

 PCR forward tag primers, PCR to tag primers, and construction of DNA sequencing libraries

 According to some embodiments of the invention, the invention provides a set of isolated PCR forward tag primers for introducing the DNA tag into a sample DNA or an equivalent thereof. According to an embodiment of the invention, each of the set of isolated PCR forward tag primers independently comprises: a first nucleic acid sequence and a second nucleic acid sequence, and the second nucleic acid sequence is linked to the 5' end of the first nucleic acid sequence, Wherein the first nucleic acid sequence is one selected from the group consisting of SEQ ID NOs: 57-62, and the second nucleic acid sequence is one selected from the group consisting of a set of isolated DNA tags according to embodiments of the invention. Herein, the expression "the second nucleic acid sequence is linked to the 5' end of the first nucleic acid sequence" means that the DNA tag selected from the embodiment according to the present invention is linked to the first nucleic acid sequence, and specifically, the DNA tag can be ligated The 5' end of a nucleic acid sequence may also be indirectly linked to the 5' end of the first nucleic acid sequence by a vector such as a linker. According to an embodiment of the present invention, the first nucleic acid sequence constituting the PCR forward tag primer has HPV specificity, and the sequence thereof is shown in Table 2 below, and will not be described herein. In the present specification, each of the set of PCR forward label primers has a DNA label according to an embodiment of the present invention as described above, and the PCR forward label can be used by PCR reaction using a PCR forward label primer. Is the primer introduced into the DNA of the sample or its equivalent, so that the corresponding DNA tag will be used? I enter DNA or its equivalent.

According to an embodiment of the invention, the invention also provides a set of isolated PCR reverse tag primers which are also used to introduce the above DNA tag into the sample DNA or its equivalent. According to an embodiment of the invention, each of the set of isolated PCR reverse tag primers independently comprises: a third nucleic acid sequence and a fourth nucleic acid sequence, and the fourth nucleic acid sequence is linked to the 3' end of the third nucleic acid sequence, Wherein the third nucleic acid sequence is one selected from the group consisting of SEQ ID NOs: 63-67, and the fourth nucleic acid sequence is one selected from the group consisting of an isolated DNA tag according to an embodiment of the present invention. Here, the expression "the fourth nucleic acid sequence is linked to the 3' end of the third nucleic acid sequence" means that the DNA tag selected from the embodiment according to the present invention is linked to the third nucleic acid sequence, and specifically, the DNA tag can be directly linked to the third The 3' end of the nucleic acid sequence may also be indirectly linked to the 3' end of the third nucleic acid sequence by a vector such as a linker. According to an embodiment of the present invention, the third nucleic acid sequence constituting the PCR reverse tag primer has HPV specificity, and the sequence thereof is shown in Table 2 below, and will not be described herein. In the present specification, the set of PCR reverse-label primers respectively have a DNA tag according to an embodiment of the present invention, and a PCR reverse-label primer can be introduced into the sample by PCR reaction using a PCR reverse-label primer. DNA or its In the equivalent, the corresponding DNA tag I is thus introduced into the DNA or its equivalent.

 According to an embodiment of the present invention, a DNA tag is introduced into a DNA sequencing library of sample DNA by a PCR amplification reaction, specifically, a PCR primer is used for a forward primer and a reverse primer, wherein the forward primer and the reverse primer are used. At least one comprising one selected from the group consisting of an isolated DNA tag according to an embodiment of the present invention, so that a DNA tag can be introduced into a DNA sequencing library of sample DNA by PCR forward tag primer and PCR reverse tag primer, thereby enabling simultaneous A DNA sequencing library of a plurality of sample DNAs was constructed. Thus, by using a PCR reaction of at least one of a PCR forward tagged substance having a DNA tag and a PCR reverse tag primer according to an embodiment of the present invention, a DNA sequencing library of a plurality of samples can be simultaneously constructed, thereby being different Different types of these tags in the DNA sequencing library distinguish between DNA sequencing libraries, and finally achieve hybrid sequencing of a large number of samples to meet the needs of high-throughput sequencing, thereby simplifying the operation process and reducing the cost of sequencing.

 In addition, the inventors have surprisingly found that when the same sample is used, based on the above method, a DNA sequencing library containing various DNA tags is constructed using different tag PCR forward tag primers or PCR reverse tag primers. The stability and reproducibility of the sequencing data results are very good.

 According to still another aspect of the present invention, the present invention also provides a kit for constructing a DNA sequencing library, the kit comprising: a set of isolated PCR forward label primers, according to an embodiment of the present invention, Each of the set of isolated PCR forward-label primers independently comprises: a first nucleic acid sequence which is one selected from the group consisting of SEQ ID NOs: 57-62; and a second nucleic acid sequence selected from the group consisting of: An example of a set of isolated DNA tags, wherein the second nucleic acid sequence is joined to the 5' end of the first nucleic acid sequence, a set of isolated PCR reverse tag primers, and a set of isolated PCR reverse tag primers An independently comprising: a third nucleic acid sequence which is one selected from the group consisting of SEQ ID NOs: 63-67; and a fourth nucleic acid sequence which is one selected from the group consisting of an isolated DNA tag according to an embodiment of the present invention Wherein the fourth nucleic acid sequence is ligated to the 3' end of the third nucleic acid sequence, wherein each of the set of isolated PCR forward tag primers and each of the set of isolated PCR reverse tag primers are separately set In different capacities In the device. Thus, with the kit, a DNA tag according to an embodiment of the present invention can be conveniently introduced into a constructed DNA sequencing library. Of course, those skilled in the art will appreciate that other components for constructing a DNA sequencing library may be included in the kit and will not be described herein.

 Method for virus detection of multiple samples

 Further, the above method for detecting a virus can be applied to a plurality of samples by means of a DNA tag. For example, in accordance with yet another aspect of the present invention, the present invention also provides a method of virus detection of a plurality of samples comprising viral nucleic acids. According to an embodiment of the invention, it comprises the following steps:

First, preparing a DNA sequencing library mixture for the plurality of samples comprising viral nucleic acids, wherein The DNA sample sequencing library mixture is composed of a DNA sequencing library of each of the plurality of samples containing viral nucleic acids, and each of the plurality of DNA samples uses a DNA tag of a different and known sequence, The DNA tag is one selected from the group consisting of SEQ ID NOs: 1-56. According to an embodiment of the present invention, the method for preparing the DNA sequencing library mixture is not particularly limited, and various DNA sequencing libraries may be separately prepared, and then mixed, or may be separately subjected to partial steps to introduce respective labels, and then mixed. Finally, the common steps of constructing a sequencing library are completed together. Thus, for the plurality of samples comprising the viral nucleic acid, preparing the DNA sequencing library mixture further comprises the steps of: separately performing each of the plurality of samples comprising the viral nucleic acid by PCR amplification reaction to obtain a plurality of expansions An increase product, wherein the PCR amplification reaction uses a forward primer and a reverse primer, the forward primer and the reverse primer are specific for the virus, and at least one of the forward primer and the reverse primer a DNA tag comprising a known sequence, the DNA tag being one selected from the group consisting of SEQ ID NOs: 1-56, and each of the plurality of samples comprising the viral nucleic acid is DNA different from each other and known in sequence Labeling; mixing the plurality of amplification products to obtain an amplification product mixture; connecting the amplification product mixture to a sequencing adaptor to obtain a ligation product mixture having a sequencing linker; and isolating and recovering the sequencing linker The ligation product mixture, the ligation product mixture with the sequencing linker, constitutes the DNA sequencing library. Optionally, according to some specific examples of the invention, the DNA sequencing library mixture for preparing the plurality of DNA samples further comprises the step of separately preparing DNA sequencing separately for each of the plurality of samples comprising viral nucleic acids a library, wherein different DNA samples are multiplexed with DNA tags of different and known sequences; and DNA sequencing libraries of the plurality of samples are combined to obtain the DNA sequencing library mixture, wherein the DNA library is Prepared by the following steps: performing a PCR amplification reaction on the sample containing the viral nucleic acid to obtain an amplification product, wherein the PCR amplification reaction uses a forward primer and a reverse primer, the forward primer and the Said reverse primer is specific for said virus; said amplification product is end-repaired to obtain an end-repaired amplification product; said end-repaired amplification product is ligated to a sequencing adaptor to obtain Sequencing the ligation product of the linker; and isolating and recovering the ligation product having the sequencing linker, which has sequencing The product constituting said connection head library DNA sequencing. Here, the term "various" is used in at least two. Wherein, the expression "different and known sequence of DNA tags" is a DNA sequencing library constructed for a sample DNA, which contains a DNA tag different from the tag of the DNA sequencing library of other sample DNA, and each tag sequence It is known that one DNA sequencing library may contain one DNA tag or two DNA tags. When it contains 1 DNA tag, the term "different" is easy to understand; when it contains 2 DNA tags, the two DNA tags can be seen as a combination, which we call "tag combination" here. The term "different from each other" means that the combination of labels contained in different DNA samples is different. According to an embodiment of the present invention, the two tags constituting the tag combination in one DNA sequencing library may be the same or different. Next, the DNA sequencing library mixture is sequenced using Ion Torrent sequencing technology to obtain viral nucleic acid sequence information of the plurality of samples and sequence information of the DNA tag.

 The viral nucleic acid sequence information is then classified based on sequence information of the DNA tag to determine the type of virus in the plurality of samples.

 The above method according to an embodiment of the present invention can make full use of high-throughput sequencing technology, for example, using Ion Torrent sequencing technology, and simultaneously sequencing DNA sequencing libraries of various sample DNAs, thereby improving sequencing efficiency and throughput of DNA sequencing libraries. At the same time, the efficiency of determining DNA sequence information of a plurality of sample DNAs can be improved. The method for sequencing and the sequencing primers used in the prior art have been described in detail above and will not be described again here.

 System for virus detection

According to still another aspect of the present invention, the present invention also provides a system for detecting a virus. Referring to FIG. 4, the system 1000 for detecting a virus includes: a DNA sequencing library preparation device 100, a sequencing device 200, and an analysis device 300, according to an embodiment of the present invention. According to an embodiment of the present invention, the DNA sequencing library preparation apparatus 100 is used for preparing a DNA sequencing library of a sample to be tested, and for example, any apparatus suitable for the DNA sequencing library construction method described above can be used as the DNA sequencing library preparation apparatus 100. The sequencing device 200 is connected to the DNA sequencing library preparation device 100, and the prepared DNA sequencing library can be received from the DNA sequencing library preparation device 100, and the received DNA sequencing library is sequenced, thereby obtaining DNA sequence information of the sample to be tested. Among them, the sequencing device is a device suitable for implementing the Ion Torrent sequencing technology. The analyzing device 300 is connected to the sequencing device 200, and can receive the obtained DNA sequence information of the sample to be tested from the sequencing device 200, thereby detecting and typing the virus in the sample to be tested based on the DNA sequence information, specifically, the DNA sequence. The information is compared with a virus database, such as an HPV database, to enable accurate virus detection and typing of the virus to be tested based on the comparison results. According to an embodiment of the present invention, the DNA sequencing library preparation apparatus may be provided with a set of isolated PCR forward label primers, each of the set of isolated PCR forward label I substances independently comprising: a first nucleic acid sequence, It is one selected from the group consisting of SEQ ID NOs: 57-62; and a second nucleic acid sequence which is one selected from the group consisting of an isolated DNA tag according to an embodiment of the present invention, wherein the second nucleic acid sequence is first A 5' end of the nucleic acid sequence is ligated, a set of isolated PCR reverse tag primers, each of the set of isolated PCR reverse tag primers independently comprising: a third nucleic acid sequence selected from the group consisting of SEQ ID NO: 63- And a fourth nucleic acid sequence which is one selected from the group consisting of an isolated DNA tag according to an embodiment of the present invention, wherein the fourth nucleic acid sequence is linked to the 3' end of the third nucleic acid sequence. Those skilled in the art will appreciate that any device known in the art suitable for performing the above operations can be utilized as a component of each of the above devices. In addition, the term "connected" as used herein is to be understood in a broad sense and may be directly connected or indirectly connected through an intermediate medium. For those skilled in the art, the specific meaning of the above terms may be understood on a case-by-case basis. The system for detecting and typing a virus according to an embodiment of the present invention can conveniently and accurately detect and classify viruses of a plurality of samples to be tested at the same time. The system has low manufacturing cost, simple and fast operation, and is beneficial to the system. It is widely promoted so that it can be applied to more women's HPV testing, so that more women can really get regular scientific and standardized screening.

 It should be noted that the method for detecting a virus according to an embodiment of the present invention and the system for detecting a virus are completed by the inventor of the present application after arduous creative labor and optimization work. The solution of the present invention will be explained below in conjunction with the embodiments. Those skilled in the art will appreciate that the following examples are merely illustrative of the invention and are not to be considered as limiting the scope of the invention. Where the specific techniques or conditions are not indicated in the examples, the techniques or conditions described in the literature in the field (for example, refer to J. Sambrook et al., Huang Peitang et al., Molecular Cloning Experimental Guide, Third Edition, Science Press) or in accordance with the product manual. The reagents or instruments used are not the manufacturer, and are commercially available products, such as those available from Life Technology.

 Example 1

 Sample

 DNA was extracted from cervical exfoliated cells from 56 known sequencing typing results using the KingFisher Automated Extractor according to the manufacturer's instructions.

 2. PCR amplification

 The 56 DNAs obtained in the sample extraction step were sequentially numbered, and 56 DNA samples obtained were amplified from 56 sets of labeled forward primers and reverse primers, respectively.

 It should be noted that different label sequences are used for different DNA samples, and the label sequences used herein are all selected from the 56 labels shown in Table 1. In addition, each of the 56 sets of labeled primers contains 6 labeled forward primers and 5 labeled reverse primers, with labeled forward primers and strips. The labeled reverse primer consists of the first nucleic acid sequence set forth in Table 2 and the tag and the third nucleic acid and tag, respectively, and the same tag selected from Table 1 is used in the same set.

 The PCR reaction was carried out in a 96-well plate, and each plate was provided with a negative control without a template, and the primer used for the negative control was the same as the primer of the sample 1.

10x Ex Taq Buffer (添加镁离子） 2.5微升 The reaction system is 25 microliters and its composition is:

10x Ex Taq Buffer (added magnesium ion) 2.5 μl

 dNTP mix (both 2.5 mM) 2 μl

 Forward primer (7.5 pmol each) 0.5 μl

 Reverse primer (7.5 pmol each) 0.5 μl

 Ex Taq HS (5U/ l) 0.125 μl

 Template DNA 5 μl

 Total volume 25 microliters

 The PCR reaction was run on a Bio-Rad PTC-200 PCR machine.

 The PCR procedure is as follows:

 95 °C 30s ^

 48 °C 30s

 72 °C 30s (40 cycles)

 72 °C 10min

 12 °C ∞

 After the completion of the PCR, 3 μl of the PCR product was detected by 2.5% agarose gel electrophoresis, and the detection result is shown in Fig. 2. According to Fig. 2, the size of the PCR product band was about 170 bp.

 3. PCR product mixing and purification

 The remaining PCR products were mixed in a 3 ml EP tube, shaken and mixed, and 500 μl of the DNA mixture was taken therefrom, purified using a Qiagen DNA Purification Kit according to the manufacturer's instructions, and the resulting 200 μl of DNA was purified. Marked as HPV-L. The DNA concentration of HPV-L was determined by Nanodrop 8000 (Thermo Fisher Scientific) to be 59.6 ng / ^ liter.

 4. End repair reaction

 The obtained sequencing library HPV-L was subjected to DNA end-repair reaction, and the reaction system for terminal repair was as follows. In this example, the Ion XpressTM Fragment Library Kit buffer and enzyme were used, and the Ion XpressTM Fragment Library Kit was referred to by reference. All instructions in this article are incorporated herein:

 Reaction system 20 (H 敫, its composition is:

 Reagent volume / reaction

 DNA fragment 39 μl

 Nuclease-free water 119 μl

5X end repair buffer 40 microliters End repair enzyme 2 μl

 Total volume 200 microliters

 The reaction conditions were as follows: using a Thermomixer (Eppendorf), a bath at 20 ° C for 20 min.

 After completion of the reaction, the purified reaction product was recovered using a QIAquick PCR purification kit, and the reaction product was dissolved in 50 μl of EB (QIAGEN Elution Buffer).

 5, 3' connector (Adapter) reaction

 Using the Ion XpressTM Fragment Library Kit, the following reaction system is used as a linker for library DNA

( adapter ) :

 The reaction system 20CH is soared and its composition is:

 The reaction conditions were as follows: using a Thermomixer (Eppendorf), a bath at 37 ° C for 30 min.

 The linker DNA sequence used is:

 P5 linker sequence:

 5, -CCACTACGCCTCCGCTTTCCTCTCTATGGGCAGTCGGTGAT

 3, -T*T*GGTGATGCGGAGGCGAAAGGAGAGATACCCGTCAGCCACTA

 A linker sequence: 5, -TTCCATCTCATCCCTGCGTGTCTCCGACTCAG

 3, -T*T*AAGGTAGAGTAGGGACGCACAGAGGCTGAGTC

 After completion of the reaction, the reaction product was recovered using 1.8-fold and Ampure Beads (Beckman Coulter Genomics), and the purified reaction product was dissolved in 40 μl of hydrazine.

 The resulting purified reaction product was subjected to electrophoresis on a 2.0% agarose gel. The condition is 100V, 2h. A 180-200 bp fragment was selected for gel extraction. Re-dissolved in 43 μl of EB to obtain a DNA fragment to which the linker was attached.

 6. Nick-translate and amplified libraries

Because there is no phosphate group at one end of the linker in the previous step, the DNA fragment to which the linker is attached needs to be gapped. The translation reaction can simultaneously amplify the DNA library. In this embodiment, the Ion Fragment Library Kit is used, and the system is as follows:

 The reaction system is:

 The reaction conditions are:

 After completion of the reaction, the reaction product was purified using 1.5 volumes of Ampure Beads (Beckman Coulter Genomics), and the purified product was dissolved in 30 μl of deionized water to obtain a DNA sequencing library.

 The obtained DNA sequencing library was detected using an Agilent 2100 Bioanalyser DNA 1000 chip (Aglient Technologies, Palo Alto, CA) and fluorescent quantitative PCR (QPCR), and the concentration results of the DNA sequencing library were 40.6 nanomolar and 38.4 nanomolar, respectively. In general, the results of the Agilent 2100 Bioanalyser DNA 1000 chip (Aglient Technologies, Palo Alto, CA) are more reliable.

 7. Preparation of Ion Torrent Sequencing Template

 7.1 Determine the appropriate library concentration

Agilent 2100 Bioanalyser DNA 1000 chip (Aglient Technologies, Palo Alto, CA). The detection results shall prevail. The DNA sequencing library prepared in the previous step is diluted to a final concentration of 560*10 ⁶ molecules per 18 μl, which is 560*10 ⁶ molecules/reaction system (280*10 ⁶ ISP). /reaction system)

 7.2 Preparation of water-in-oil emulsion

IKA DT-20 oil phase (9 ml), Ion SphereTM particles, DNA sequencing library, and PCR aqueous phase MIX were prepared according to the Ion XpressTM Template Kit instructions. The composition of the PCR aqueous phase MIX is:

 Finally, the diluted DNA sequencing library (18^:L/reaction) was mixed with PCR aqueous phase MIX for emulsion PCR reaction.

 The reaction procedure is as follows:

 Through the above emulsion PCR reaction, DNA molecules in the DNA sequencing library were ligated and replicated with Ion SphereTM particles, and the structure diagram after the connection is shown in FIG. As shown in Fig. 3, the 5' end and the 3' end of the DNA fragment are linked to the linker 1 and the linker 2, respectively, and are connected to the ion magnetic particles through the linker 2. The sequences of Connector 1 and Connector 2 are as follows:

Name sequence

Connector 1 (A connector) TTCCATCTCATCCCTGCGTGTCTCCGACTCAG

Connector 2 (magnetic beads - P5 CCTATCCCCTGTGTGCCTTGGCAGTCTCAG-CCTCTCTATGGGCA connector) GTCGGTGAT 7.3 Preparation of Ion SphereTM particles carrying single-stranded DNA

 After the completion of the emulsion PCR, the Ion SphereTM particles were separated and recovered, and Ion Sphere was passed through with the lysate (the aqueous solution containing NaOH and Tween-20, the final concentrations of NaOH and Tween-20 were 125 mM and 0.1% by weight, respectively). The DNA template on the TM particles changes from double stranded to single stranded.

 Then, using MyOneTM beads with biotin, Ion SphereTM particles carrying single-stranded DNA are enriched by specific binding of MyOneTM beads to the amplified product, thereby obtaining a sequencing template for Ion Torrent sequencing technology. .

 Qualified by Qubit 2.0 (Invitrogen) to meet the sequencing requirements of the machine.

 6. Ion Torrent sequencing

 The sequencing procedure is detailed in the Ion Torrent operating instructions. The corresponding sequencing chip is installed. In the embodiment, the 314 chip is used, and the enzyme and the prepared Ion SphereTM particle carrying the single-stranded DNA are added to the chip for sequencing, and the sequencing process can be completed in about 2.5 hours to obtain a reliable gene. Sequence information.

 7. Analysis of results

 By screening the sequence of the tag in the sequencing result and the sequence information of the primer, the DNA sequence information of each sample can be obtained, and the obtained DNA sequence information is compared with the HPV database, and finally the HPV detection and typing of each sample can be realized. The results obtained are completely consistent with the original known results. The specific results are as follows:

 Sample sample Original sequencing test This test result is the original sequencing test result.

 Number number result

 1 HPV16, HPV56 HPV16, HPV56 29 HPV18 HPV18

 HPV18, HPV31, HPV18, HPV31, negative negative

2 30

 HPV39 HPV39

 3 negative negative 31 negative negative

 4 HPV45, HPV11 HPV45, HPV11 32 HPV16 HPV16

 5 HPV56, HPV31 HPV56, HPV31 33 HPV18 HPV18

 6 HPV68 HPV68 34 negative negative

 HPV18, HPV18,

7 HPV39, HPV18 HPV39, HPV18 35 HPV11, HPV11,

 HPV16 HPV16

 8 HPV56 HPV56 36 HPV66 HPV66

 9 HPV45 HPV45 37 HPV18 HPV18

10 HPV35 HPV35 38 HPV58 HPV58 11 HPV52 HPV52 39 HPV33 HPV33

 12 HPV66 HPV66 40 HPV31 HPV31

 13 HPV18, HPV16 HPV18, HPV16 41 HPV16 HPV16

 14 HPV11 HPV11 42 HPV68 HPV68

 15 HPV18, HPV31 HPV18, HPV31 43 HPV52 HPV52

 16 negative negative 44 HPV90 HPV90

 HPV39 HPV39 HPV18, HPV18,

17 45

 HPV31 HPV31

 18 HPV51 HPV51 46 HPV45 HPV45

 19 HPV11 HPV11 47 negative negative

 20 negative negative 48 HPV6 HPV6

 21 HPV18, HPV16 HPV18, HPV16 49 HPV16 HPV16

 HPV51 HPV51 HPV18, HPV18,

22 50

 HPV31 HPV31

 23 HPV39 HPV39 51 HPV31 HPV31

 24 HPV52 HPV52 52 HPV56 HPV56

 25 HPV18, HPV6 HPV18, HPV6 53 HPV45 HPV45

 26 HPV90 HPV90 54 HPV45 HPV45

 27 HPV33 HPV33 55 HPV51 HPV51

 28 HPV18 HPV18 56 HPV6, HPV33 HPV6, HPV33 As shown in the above table, using the technical route of the present invention, HPV genotyping of samples of 56 known sequencing typing results revealed that: the technical solution of the present invention was used. The virus, ie HPV, was analyzed, and the obtained results were consistent with the known typing results, thereby demonstrating that the technical scheme of the present invention can be effectively applied to the detection of viruses, especially HPV, and compared with existing sequencing types. Methods, the technical methods of the present invention are much faster. Further, as shown in the above table, the technical solution of the present invention can be effectively applied to various types of viruses, that is, it is possible to effectively detect a plurality of HPV types.

Industrial applicability

 DNA tag, PCR forward tag primer, PCR reverse tag I, DNA sequencing library and preparation method thereof, kit for constructing DNA sequencing library, and DNA sample for constructing DNA sequencing library of the present invention

Method of DNA sequence information, method of determining DNA sequence information of a plurality of DNA samples, and performing HPV on a sample The method of typing, the method of typing HPV for multiple samples, and the system for detecting and typing HPV can be applied to HPV detection and classification, which can effectively improve the HPV detection of multiple samples to be tested. The efficiency of the type, and the results are accurate, the cost is low, and the operation is simple and fast. Although specific embodiments of the invention have been described in detail, those skilled in the art will understand. Various modifications and substitutions may be made to those details in light of the teachings of the invention, which are within the scope of the invention. The full scope of the invention is given by the appended claims and any equivalents thereof.

 In the description of the present specification, the description of the terms "one embodiment", "some embodiments", "illustrative embodiment", "example", "specific example", or "some examples", etc. Particular features, structures, materials or features described in the examples or examples are included in at least one embodiment or example of the invention. In the present specification, the schematic representation of the above terms does not necessarily mean the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in a suitable manner in any one or more embodiments or examples.

Claims

Claim  A virus detection method, comprising the steps of:  Preparing a DNA sequencing library for a sample containing viral nucleic acid;  DNA sequencing of the DNA sequencing library using Ion Torrent technology to obtain sequence information of the viral nucleic acid;  The type of the virus is determined based on sequence information of the viral nucleic acid.  The virus detecting method according to claim 1, wherein the DNA sequencing library is prepared by the following steps:  The sample containing the viral nucleic acid is subjected to a PCR amplification reaction to obtain an amplification product, wherein the PCR amplification reaction uses a forward primer and a reverse primer, and the forward primer and the reverse primer are Said virus-specific; end-repairing said amplification product to obtain an end-repaired amplification product;  The end-repaired amplification product is ligated to a sequencing linker to obtain a ligation product having a sequencing linker;  The ligation product having the sequencing linker is isolated and recovered, and the ligation product having the sequencing linker constitutes the DNA sequencing library.  The virus detecting method according to claim 2, wherein the virus is a human papillomavirus. The virus detecting method according to claim 3, wherein the sample containing the viral nucleic acid is a whole-genome DNA sample of a female biological sample.  The virus detecting method according to claim 4, wherein the female biological sample is at least one selected from the group consisting of female cervical exfoliated cells and cervical cancer tissues.  The virus detecting method according to claim 5, wherein the whole genomic DNA sample is carried out using a KingFisher automatic extractor.  The virus detection method according to claim 2, wherein the forward primer comprises a first nucleic acid sequence 'J, and the first nucleic acid sequence is one selected from the group consisting of SEQ ID NOs: 57-62 .  The virus detection method according to claim 7, wherein the forward primer further comprises a second nucleic acid sequence, and the second nucleic acid sequence is one selected from the group consisting of SEQ ID NOs: 1-56.  Its towel,  The second nucleic acid sequence is joined to the 5' end of the first nucleic acid sequence.  The virus detecting method according to claim 2, wherein the reverse primer comprises a third nucleic acid sequence, and the third nucleic acid sequence is one selected from the group consisting of SEQ ID NOs: 63-67. The virus detection method according to claim 9, wherein the reverse primer further comprises a fourth nucleic acid sequence, and the fourth nucleic acid sequence is one selected from the group consisting of SEQ ID NOs: 1-56. Its towel,  The fourth nucleic acid sequence is joined to the 3' end of the third nucleic acid sequence.  The virus detecting method according to claim 2, further comprising the step of purifying the amplification product before performing the terminal repair on the amplification product.  The virus detecting method according to claim 2, further comprising the step of purifying the terminal-repaired amplification product before the terminal-repaired amplification product is linked to the sequencing adaptor. .  The virus detecting method according to claim 2, wherein the ligation product having the sequencing linker has a length of about 180 to 200 bp.  The virus detecting method according to claim 2, wherein the ligation product having the sequencing linker is separated and recovered by electrophoresis at 100 V for 2 hours using a 2.0% agarose gel.  The virus detecting method according to claim 2, further comprising performing a nick translation reaction of the ligation product having the sequencing linker.  The virus detecting method according to claim 15, further comprising performing a PCR amplification reaction on the ligation product subjected to the nick translation.  17. The method of claim 1, wherein sequencing the DNA sequencing library of the DNA sample further comprises the step of preparing an Ion Torrent sequencing template by an emulsion PCR reaction.  18. The method of claim 17, wherein preparing the Ion Torrent sequencing template by emulsion PCR reaction further comprises the steps of:  Forming a water-in-oil emulsion, the water-in-oil emulsion comprising a plurality of discrete aqueous compartments, at least a portion of the plurality of discrete aqueous compartments comprising:  a magnetic particle, a portion of the DNA sequencing library, an oligonucleotide primer for amplifying the DNA sequencing library, wherein at least a portion of the oligonucleotide primer is linked to the magnetic particle;  The water-in-oil emulsion is placed under thermal cycling conditions suitable for amplifying the DNA sequencing library to form magnetic particles on which the DNA sequencing library is carried;  The magnetic particles carrying the DNA sequencing library thereon are placed under conditions suitable for double-stranded DNA cleavage to obtain magnetic particles carrying single-stranded DNA thereon, and the magnetic particles carrying the single-stranded DNA thereon constitute an Ion Torrent sequencing template .  19. The method of claim 18, wherein each of said plurality of discrete aqueous compartments contains at most one DNA molecule of said DNA sequencing library.  20. The method of claim 18, wherein the magnetic particles are Ion SphereTM particles. 21. The method according to claim 18, wherein the oligonucleotide primer for amplifying the DNA sequencing library comprises a first oligonucleotide primer and a second oligonucleotide primer, Its towel,  The sequence of the first oligonucleotide primer is TTCCATCTCATCCCTGCGTGTCTCCGACTCAG, and the first oligonucleotide primer is linked to the magnetic particle;  The sequence of the second oligonucleotide primer is CCTATCCCCTGTGTGCCTTGGCAGTCTCAG-CCTCTCTATGGGCAGTCGGTGAT, and the second oligonucleotide primer is in a free state in the aqueous compartment.  22. The method of claim 18, wherein the thermal cycling conditions suitable for amplifying the DNA sequencing library comprise the following:  Step 1: 94 °C 6min  Step 2: 94 °C 30 sec  Step 3: 58 °C 30 sec  Step 4: 72 °C 90sec  Repeat steps 2-4, 40 cycles; and  Step 5: 94 °C 30 sec  Step 6: 68 °C 6min  Repeat steps 5 and 6, 5 cycles.  23. The method according to claim 18, wherein the water-in-oil emulsion is placed under thermal cycling conditions suitable for amplifying the DNA sequencing library to liquefy the water-in-oil emulsion The liquid contact, wherein the lysate contains an aqueous solution of NaOH and Tween-20, the final concentrations of the NaOH and Tween-20 being 125 mM and 0.1% by weight, respectively.  24. The method of claim 18, further comprising enriching the magnetic particles carrying the single-stranded DNA thereon to obtain the Ion Torrent sequencing template.  The method according to claim 24, wherein the enrichment of the magnetic particles carrying the single-stranded DNA thereon is carried out by using MyOneTM beads.  26. The method of claim 17, further comprising the step of diluting the DNA sequencing library prior to performing the emulsion PCR amplification. The method according to claim 26, wherein in the emulsion PCR amplification reaction system, the concentration of the ligation product having the sequencing linker is 560*10 ⁶ molecules/reaction system.  28. A system for virus detection, comprising:  a DNA sequencing library preparation device for preparing a DNA sequencing library against a sample containing viral nucleic acid; a sequencing device, the sequencing device being coupled to the DNA sequencing library preparation device for sequencing the DNA The library is sequenced to obtain sequence information of the viral nucleic acid;  An analysis device, the analysis device being coupled to the sequencing device, configured to determine a type of the virus based on sequence information of the viral nucleic acid,  Wherein the sequencing device is a device suitable for implementing the Ion Torrent sequencing technology.