KR102827389B1 - Kit and method for differential diagnosis of Bovine viral diarrhea virus subgenotype - Google Patents

Abstract

The present invention relates to a composition, kit or method for differential diagnosis of bovine viral diarrhea virus genotypes. When the composition, kit or method of the present invention is used, it exhibits the effect of being able to distinguish and detect bovine viral diarrhea virus genotypes with high sensitivity and specificity through successive PCR reactions.

Description

Kit and method for differential diagnosis of Bovine viral diarrhea virus subgenotype

The present invention relates to a simultaneous genetic subtype differentiation diagnostic kit and diagnostic method for bovine viral diarrhea virus (BVDV). More specifically, the present invention relates to a real-time genetic diagnostic kit and diagnostic method capable of differentiating between types 1 and 2, and types 1a and 1b.

Bovine viral diarrhea (BVD) is a representative productivity-reducing disease in cattle, causing diarrhea and pseudo-acidosis, and it continuously causes great damage to farms. Assuming that the domestic cattle farm size is 2 million, the annual loss is estimated to be at least 100 billion won (KRW 50,000-60,000/head/year). For this reason, although it is a non-statutory contagious disease existing in Korea, we are continuously promoting farm monitoring and promotion through livestock quarantine projects and research projects. As of now, 17 BVDV-1 subgenotypes and 4 BVDV-2 subgenotypes have been reported worldwide, so the genotypes are diverse, and new genotypes are being reported due to continuous mutation. Domestic BVDV genetic analysis results showed that most of them belong to the BVDV type 1a and type 2a groups, and recently, the occurrence of BVDV 1b type has increased significantly more than BVDV 1a type, and rarely, BVDV 1n and 1o types have also been reported. The present inventors intend to contribute to public health by establishing a rapid and accurate diagnosis system for bovine viral diarrhea virus.

The present inventors have made extensive research efforts to develop a diagnostic kit capable of simultaneously differentiating multiple genotypes of bovine viral diarrhea virus based on rapid and accurate diagnosis, in order to minimize economic losses to small-scale farmers due to bovine viral diarrhea virus, and to contribute to livestock quarantine and public health. As a result, they have confirmed that the differential diagnosis kit for bovine viral diarrhea virus of the present invention enables rapid and accurate differential diagnosis of bovine viral diarrhea virus genotypes, and have completed the present invention.

Accordingly, the purpose of the present invention is to provide a composition for differential diagnosis of bovine viral diarrhea virus (BVDV) genotypes.

Another object of the present invention is to provide a kit for differential diagnosis of bovine viral diarrhea virus (BVDV) genotypes.

Another object of the present invention is to provide a method for differential diagnosis of bovine viral diarrhea virus (BVDV) genotypes.

According to one aspect of the present invention, the present invention provides a composition for differential diagnosis of bovine viral diarrhea virus genotypes, comprising a primer pair for detecting bovine viral diarrhea virus (BVDV) genotype 1 or 2 consisting of nucleotide sequences of SEQ ID NOs: 1 and 2.

The term "primer" as used herein means an oligonucleotide capable of acting as an initiation point of synthesis under conditions that induce synthesis of a primer extension product complementary to a nucleic acid strand (template), i.e., in the presence of nucleotides and a polymerization agent such as DNA polymerase, and at a suitable temperature and pH.

In one embodiment of the present invention, the composition additionally comprises a BVDV type 1 differential diagnosis probe comprising a nucleotide sequence of SEQ ID NO: 3; a BVDV type 2 differential diagnosis probe comprising a nucleotide sequence of SEQ ID NO: 4; or a combination thereof.

The term "probe" as used herein means a single-stranded nucleic acid molecule comprising a portion or portions substantially complementary to a target nucleic acid sequence. The "target nucleic acid", "target nucleic acid sequence" or "target sequence" means a nucleic acid sequence to be detected, which anneals or hybridizes with a primer or probe under hybridization, annealing, or amplification conditions.

More specifically, the probe and primer are single-stranded deoxyribonucleotide molecules. The probe or primer used in the present invention may include naturally occurring dNMPs (i.e., dAMP, dGMP, dCMP and dTMP), modified nucleotides or non-natural nucleotides. Additionally, the probe or primer may include ribonucleotides.

The primer must be long enough to prime the synthesis of the extension product in the presence of the polymerization agent. The exact length of the primer will depend on several factors, including, for example, temperature, application, and the source of the primer.

The term "annealing" or "priming" as used herein means the apposition of an oligodeoxynucleotide or nucleic acid to a template nucleic acid, which causes a polymerase to polymerize the nucleotides to form a nucleic acid molecule complementary to the template nucleic acid or a portion thereof.

The primer used in the present invention hybridizes or anneals to a portion of the template to form a double-stranded structure. Conditions for nucleic acid hybridization suitable for forming such a double-stranded structure are disclosed in Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001) and Haymes, B. D., et al., Nucleic Acid Hybridization, A Practical Approach, IRL Press, Washington, D.C. (1985).

The term "hybridization" as used herein refers to the formation of a double-stranded nucleic acid from complementary single-stranded nucleic acids. Hybridization can occur between two nucleic acid strands that are completely identical or substantially identical with some mismatch. The complementarity for hybridization can vary depending on the hybridization conditions, particularly temperature. The terms "annealing" and "hybridization" as used herein are not different and are used interchangeably herein.

In one embodiment of the present invention, the 5'-terminal of the nucleotide sequence of SEQ ID NO: 3 or SEQ ID NO: 4 is labeled with one different fluorophore selected from the group consisting of FAM, TET, HEX, JOE, ROX, TMR, Cy5, TxR, and Cal Red 610, and the 3'-terminal is modified with a quencher of BHQ-1, BHQ-2 or BHQ-3, but is not limited thereto.

According to another aspect of the present invention, the present invention provides a composition for differential diagnosis of bovine viral diarrhea virus genotypes, comprising (i) a primer pair for detecting bovine viral diarrhea virus (BVDV) genotype 1 or 2 consisting of the nucleotide sequences of SEQ ID NOs: 1 and 2; and (ii) a primer pair for detecting BVDV genotype 1a or 1b consisting of the nucleotide sequences of SEQ ID NOs: 5 and 6.

In one embodiment of the present invention, the composition is a composition additionally comprising:

(i) a probe for differential diagnosis of BVDV type 1 consisting of a nucleotide sequence of sequence number 3; a probe for differential diagnosis of BVDV type 2 consisting of a nucleotide sequence of sequence number 4; or a combination thereof,

(ii) a probe for differential diagnosis of BVDV type 1a consisting of a nucleotide sequence of sequence number 7; a probe for differential diagnosis of BVDV type 1a consisting of a nucleotide sequence of sequence number 8; a probe for differential diagnosis of BVDV type 1b consisting of a nucleotide sequence of sequence number 9; a probe for differential diagnosis of BVDV type 1b consisting of a nucleotide sequence of sequence number 10, or

(iii) combinations of these.

The primers and probes of the above sequence numbers 1 to 4 target the 5'UTR gene of BVDV belonging to BVDV genotype 1 or 2.

The primers and probes of the above sequence numbers 5 to 10 target the 5'UTR gene of BVDV belonging to BVDV genotypes 1a and 1b.

In one embodiment of the present invention, the 5'-terminal of a probe consisting of any one of the nucleotide sequences of SEQ ID NOs: 7 to 10 is labeled with a fluorophore selected from the group consisting of FAM, TET, HEX, JOE, ROX, TMR, Cy5, TxR, and Cal Red 610, and the 3'-terminal is modified with a quencher of BHQ-1, BHQ-2 or BHQ-3, but is not limited thereto.

In another embodiment of the present invention, the probe comprising the nucleotide sequence of SEQ ID NO: 7 and the probe comprising the nucleotide sequence of SEQ ID NO: 8 are labeled with one or more fluorescent substances which are the same or different from each other.

In another embodiment of the present invention, the probe comprising the nucleotide sequence of SEQ ID NO: 9 and the probe comprising the nucleotide sequence of SEQ ID NO: 10 are labeled with at least one fluorescent substance which is the same as or different from each other.

According to another aspect of the present invention, the present invention provides a kit for differential diagnosis of bovine viral diarrhea virus genotypes comprising the composition according to one aspect of the present invention.

Since the kit of the present invention includes the composition as a component, the contents commonly applied to both inventions are equally applied to the kit invention. In addition, in order to avoid duplicate descriptions in this specification, common elements between both inventions will be omitted.

According to another aspect of the present invention, the present invention provides a method for differential diagnosis of bovine viral diarrhea virus genotypes, comprising the following steps:

(a) a step of preparing nucleic acid from a specimen collected from a cow or the environment;

(b) a step of amplifying the target nucleotide sequence in the test sample using (i) a composition of any one of claims 1 to 3; or (ii) a composition of any one of claims 4 to 8; and

(c) A step of confirming the above amplification results.

In one embodiment of the present invention, the amplification is performed by PCR (polymerase chain reaction).

The polymerase chain reaction (PCR) described above is the best-known nucleic acid amplification method, and many modifications and applications have been developed. For example, touchdown PCR, hot start PCR, nested PCR, and booster PCR have been developed by modifying the traditional PCR procedure to enhance the specificity or sensitivity of PCR. In addition, real-time PCR, differential display PCR (DD-PCR), rapid amplification of cDNA ends (RACE), multiplex PCR, inverse polymerase chain reaction (IPCR), vectorette PCR, thermal asymmetric interlaced PCR (TAIL-PCR), and multiplex PCR have been developed for specific applications. For more information on PCR, see McPherson, M.J., and Moller, S.G. PCR. BIOS Scientific Publishers, Springer-Verlag New York Berlin Heidelberg, N.Y. (2000), whose teachings are incorporated herein by reference.

In one embodiment of the present invention, the amplification is performed by real-time PCR.

In one embodiment of the present invention, the real-time PCR is performed using the TaqMan probe method.

The kit of the present invention performs multiplex detection (or multiplexing detection) of two or more types of bovine viral diarrhea viruses simultaneously through a polymerase chain reaction method using the above primers and probes.

The above “multiplex detection” or “multiplexing detection” refers to the simultaneous detection of multiple target nucleic acid sequences in a reaction vessel (e.g., a reaction tube).

The term "multiplex PCR" as used herein means that multiple targets are amplified simultaneously by polymerase chain reaction in a reaction vessel.

Various DNA polymerases can be utilized in the above polymerase chain reaction, including the 'Klenow' fragment of E. coli DNA polymerase I, thermostable DNA polymerase, and bacteriophage T7 DNA polymerase. Specifically, the polymerase is a thermostable DNA polymerase obtainable from various bacterial species, including Thermus aquaticus (Taq), Thermus thermophilus (Tth), Thermus filiformis , Thermis flavus , Thermococcus literalis , and Pyrococcus furiosus (Pfu).

According to one embodiment of the present invention, the kit of the present invention comprises Taq DNA polymerase or Pfu DNA polymerase.

According to another embodiment of the present invention, the kit of the present invention comprises Taq DNA polymerase.

The kit of the present invention may additionally contain dUTP (deoxyuridine triphosphate) and UDG (Uracil DNA Glycosilase) to block carryover or crossover contamination by polymerase chain reaction products. UDG recognizes and cleaves uracil included in the DNA template strand of the previous amplification product, and the cleaved DNA template strand no longer functions as a template strand, thereby preventing the amplification reaction from occurring. The present invention uses dUTP and UDG to fundamentally block the generation of amplification products due to contamination of the previous amplification product, thereby excluding false positive results due to laboratory contamination of the previous amplification product, thereby improving the accuracy of the test.

When conducting a polymerization reaction, it is desirable to provide the components required for the reaction in excess in the reaction vessel. The excess of the components required for the amplification reaction means an amount such that the amplification reaction is not substantially limited by the concentration of the components. It is desirable to provide cofactors such as Mg ²⁺ , dATP, dCTP, dGTP and dTTP to the reaction mixture in an amount such that the desired degree of amplification can be achieved. All the enzymes used in the amplification reaction can be active under the same reaction conditions. In fact, the buffer allows all the enzymes to approach the optimal reaction conditions. Therefore, the amplification process of the present invention can be performed in a single reaction without changing the conditions such as adding a reactant.

In the present invention, annealing is performed under stringent conditions that enable specific binding between the target nucleotide sequence and the primer. Stringent conditions for annealing are sequence-dependent and vary depending on surrounding environmental variables.

In one embodiment of the present invention, the gene amplification is performed by real-time PCR.

The above real-time PCR is a technology for monitoring and analyzing the increase of PCR amplification products in real time (Levak KJ, et al. , PCR Methods Appl. , 4(6): 357-62(1995)). The PCR reaction can be monitored by recording the fluorescence emission at each cycle during the exponential phase, where the increase of PCR products is proportional to the initial amount of target template. The higher the starting copy number of the nucleic acid target, the faster the fluorescence increase is observed and the lower the Ct value (cycle threshold). A marked increase in fluorescence above the reference value measured between 3 and 15 cycles indicates the detection of accumulated PCR products. Compared to conventional PCR methods, real-time PCR has the following advantages: (a) whereas conventional PCR is measured at a plateau, real-time PCR can obtain data during the exponential growth phase; (b) the increase in reporter fluorescence signal is directly proportional to the number of generated amplicons; (c) the digested probe provides permanent record amplification of the amplicon; (d) an increased detection range; (e) more than 1,000 times less nucleic acids are required compared to conventional PCR methods; (f) detection of the amplified DNA is possible without separation by electrophoresis; (g) increased amplification efficiency can be obtained by utilizing small amplicon sizes; and (h) there is a reduced risk of contamination.

When the PCR amplification product amount reaches a detectable amount by fluorescence, an amplification curve begins to occur, and the signal increases exponentially before reaching a plateau. The larger the initial DNA amount, the fewer the number of cycles for the amplification product amount to reach a detectable amount, so the amplification curve appears quickly. Therefore, when a real-time PCR reaction is performed using a stepwise diluted standard sample, an amplification curve is obtained that is lined up at equal intervals in order of initial DNA amount. Here, if the threshold is set at an appropriate point, the Ct value at which the threshold and the amplification curve intersect is calculated.

In real-time PCR, PCR amplification products are detected through fluorescence. Detection methods include the interchelating method (SYBR Green I method) and the method using a fluorescently labeled probe (TaqMan probe method). Since the interchelating method detects all double-stranded DNA, there is no need to prepare probes for each gene, so the reaction system can be constructed at low cost. The method using a fluorescently labeled probe is expensive, but has high detection specificity, so even similar sequences can be distinguished and detected. According to an embodiment of the present invention, the kit of the present invention uses the TaqMan probe method.

First, the interchelating method is a method that utilizes a double-stranded DNA binding dye, and quantifies non-specific amplification and amplicon production including primer-dimer complexes using a non-sequence-specific fluorescent intercalating reagent (SYBR Green I or ethidium bromide). The reagent does not bind to ssDNA. SYBR Green I is a fluorescent dye that binds to the minor groove of double-stranded DNA, and is a reagent (interchelator) that shows almost no fluorescence in solution but exhibits strong fluorescence when bound to double-stranded DNA (Morrison TB, Biotechniques. , 24(6): 954-8, 960, 962(1998)). Therefore, the amount of amplification product produced can be measured because fluorescence is emitted through the binding between SYBR Green I and double-stranded DNA. SYBR Green real-time PCR is accompanied by an optimization process such as melting point or dissociation curve analysis for amplicon identification. Normally, SYBR Green is used in singleplex reactions, but it can be used in multiplex reactions when accompanied by melting curve analysis (Siraj AK, et al. , Clin Cancer Res. , 8(12): 3832-40(2002); and Vrettou C., et al. , Hum Mutat. , Vol 23(5): 513-521(2004)).

The Ct (cycle threshold) value is the cycle number at which the fluorescence generated in the reaction exceeds the threshold, and is inversely proportional to the logarithm of the initial copy number. Therefore, the Ct value assigned to a particular well reflects the number of cycles at which a sufficient number of amplicons has accumulated in the reaction. The Ct value is the cycle at which an increase in △Rn is first detected. Rn represents the magnitude of the fluorescence signal generated during PCR at each time point, and △Rn represents the fluorescence emission intensity of the reporter die divided by the fluorescence emission intensity of the reference die (normalized reporter signal). The Ct value is also called the Cp (crossing point) in LightCycler. The Ct value represents the point at which the system begins to detect an increase in the fluorescence signal associated with the exponential growth of the PCR product in the log-linear phase. This period provides the most useful information about the reaction. The slope of the log-linear phase represents the amplification efficiency (Eff) (www.appliedbiosystems.co.kr/).

Meanwhile, TaqMan probes are typically longer oligonucleotides (e.g., 20-30 nucleotides) than the primers that contain a fluorophore at the 5'-end and a quencher (e.g., TAMRA or non-fluorescent quencher (NFQ)) at the 3'-end. The excited fluorophore transfers energy to a nearby quencher rather than fluorescing (FRET = FRET or fluorescence resonance energy transfer; Chen, X., et al. , Proc Natl Acad Sci USA , 94(20): 10756-61(1997)). Therefore, when the probe is normal, no fluorescence is emitted. TaqMan probes are designed to anneal to an internal region of a PCR product.

TaqMan probe specifically hybridizes to template DNA in the annealing step, but fluorescence is suppressed by a quencher on the probe. During the extension reaction, the TaqMan probe hybridized to the template is degraded by the 5' to 3' nuclease activity of Taq DNA polymerase, releasing the fluorescent dye from the probe, thereby releasing the suppression by the quencher and causing fluorescence. At this time, the 5'-terminus of the TaqMan probe must be located downstream of the 3'-terminus of the extension primer. That is, when the 3'-terminus of the extension primer is extended by template-dependent nucleic acid polymerase, the 5'-terminus of the TaqMan probe is cleaved by the 5' to 3' nuclease activity of the polymerase, thereby generating a fluorescence signal of the reporter molecule.

The reporter molecule and quencher molecule coupled to the TaqMan probe include a fluorescent substance and a non-fluorescent substance.

Any fluorescent reporter molecule and quencher molecule that can be used in the present invention can be used as known in the art, and examples thereof are as follows (the numbers in parentheses are the maximum emission wavelengths in nanometers): Cy2 ^TM (506), YOPRO ^TM -1 (509), YOYO ^TM -1 (509), Calcein (517), FITC (518), FluorX ^TM (519), Alexa ^TM (520), rhodamine 110 (520), 5-FAM (522), Oregon Green ^TM 500 (522), Oregon Green ^TM 488 (524), RiboGreen ^TM (525), RhodamineGreen ^TM (527), Rhodamine 123 (529), Magnesium Green ^TM (531), Calcium Green ^TM (533), TO-PRO ^TM -1 (533), TOTO1 (533), JOE(548), BODIPY530/550(550), Dil(565), BODIPY TMR(568), BODIPY558/568(568), BODIPY564/570(570), Cy3 ^TM (570), Alexa ^TM 546(570), TRITC(572), Magnesium Orange ^TM (575), Phycoerythrin R&B (575), Rhodamine Phalloidin (575), Calcium Orange ^TM (576), Pyronin Y (580), RhodamineB (580), TAMRA (582), Rhodamine Red ^TM (590), Cy3.5 ^TM (596), ROX (608), Calcium Crimson ^TM (615), Alexa ^TM 594(615), Texas Red(615), Nile Red(628), YO-PRO ^TM -3(631), YOYO ^TM -3(631), R-phycocyanin(642), CPhycocyanin(648), TO-PRO ^TM -3(660), TOTO3(660), DiD DilC(5)(665), Cy5 ^TM (670), Thiadicarbocyanine(671), Cy5.5(694), HEX(556), TET(536), VIC(546), BHQ-1(534), BHQ-2(579), BHQ-3(672), BiosearchBlue(447), CAL Fluor Gold 540(544), CAL Fluor Orange 560(559), CAL Fluor Red 590(591), CAL FluorRed 610(610), CAL Fluor Red 635(637), FAM (520), Fluorescein (520), Fluorescein-C3 (520), Pulsar 650 (566), Quasar 570 (667), Quasar 670 (705), Quasar 705 (610) and TxR (592).

Suitable reporter-quencher pairs are described in many references: Pesce et al., editors, FLUORESCENCE SPECTROSCOPY (Marcel Dekker, New York, 1971); White et al., FLUORESCENCE ANALYSIS: A PRACTICAL APPROACH (Marcel Dekker, New York, 1970); Berlman, HANDBOOK OF FLUORESCENCE SPECTRA OF AROMATIC MOLECULES, 2nd EDITION (Academic Press, New York, 1971); Griffiths, COLOUR AND CONSTITUTION OF ORGANIC MOLECULES (Academic Press, New York, 1976); Bishop, editor, INDICATORS (Pergamon Press, Oxford, 1972); Haugland, HANDBOOK OF FLUORESCENT PROBES AND RESEARCH CHEMICALS (Molecular Probes, Eugene, 1992); Pringsheim, FLUORESCENCE AND PHOSPHORESCENCE (Interscience Publishers, New York, 1949); Haugland, R. P., HANDBOOK OF FLUORESCENT PROBES AND RESEARCH CHEMICALS, Sixth Edition, Molecular Probes, Eugene, Oreg., 1996; U.S. Pat. Nos. 3,996,345 and 4,351,760.

In one embodiment of the present invention, the probe has a label bound thereto, and the confirmation in step (c) is performed by detecting a signal generated from the probe.

The above method is explained step by step below.

(a) Step of preparing nucleic acid from a specimen

The term "specimen sample" in this specification includes various samples. More specifically, the specimen sample may be a sample isolated from a subject (cattle) suspected of being infected with the virus, but is not limited thereto, and may be an environmental sample isolated from the surrounding environment of the subject (cattle). The sample isolated from the subject may be a cell, tissue, feces, urine, or body fluid, and the body fluid may be blood, serum, plasma, lymph, ocular fluid, saliva, or tissue extract, but is not limited thereto. The environmental sample may include, but is not limited to, litter, drinking water, feed, etc.

The step of preparing nucleic acids from the above specimen sample is a step of processing the specimen sample to apply the PCR method. The processing method of the above specimen sample may apply a DNA extraction method or an RNA extraction method, and more specifically, a DNA extraction method may be applied. Extracting DNA or RNA from the sample may be performed according to a conventional method known in the art (references: Sambrook, J. et al., Molecular Cloning. A Laboratory Manual, 3rd ed. Cold Spring Harbor Press (2001); Tesniere, C. et al., Plant Mol. Biol. Rep., 9:242 (1991); Ausubel, F.M. et al., Current Protocols in Molecular Biology, John Willey & Sons (1987); and Chomczynski, P. et al., Anal. Biochem. 162:156 (1987)).

(b) a step of amplifying the sequence using the primer pair and probe described above;

In one embodiment of the present invention, the amplification is performed by PCR (polymerase chain reaction).

In another embodiment of the present invention, the amplification is performed by real-time PCR.

In one specific embodiment of the present invention, the real-time PCR is performed using the TaqMan probe method.

In one embodiment of the present invention, the probe has a label bound thereto, and the identification in step (c) is performed by detecting a signal generated from the probe.

Since the method of the present invention uses the kit for differential diagnosis of bovine viral diarrhea virus genotypes, the common content between the two is omitted to avoid excessive complexity of this specification.

(c) A step of confirming the above amplification results.

When amplifying a sequence using the traditional PCR method, the amplification result can be confirmed by agarose gel or polyacrylamide gel electrophoresis, and more specifically, by agarose gel electrophoresis, but is not limited thereto. After electrophoresis, the electrophoresis result can be analyzed by ethidium bromide staining, etc.

When a sequence is amplified using the real-time PCR method, regardless of whether the unknown sample is positive or negative, the change in the fluorescence intensity of the label over the entire PCR amplification period of the internal control is measured, the number of PCR reactions (Cp value or Ct value) that reach a certain fluorescence intensity is analyzed, and a standard curve of fluorescence intensity can be obtained. This standard curve of the Cp value or Ct value and fluorescence intensity of the internal control is clearly distinguishable from the curve pattern of the Cp value or Ct value and fluorescence intensity of the negative control, and can provide a criterion for determining whether the unknown sample is positive or negative. Therefore, by PCR amplifying a specimen sample, measuring the change in the fluorescence intensity of the label over the entire PCR amplification period, analyzing the number of PCR reactions (Cp value or Ct value) that reach a certain fluorescence intensity, and obtaining a standard curve of fluorescence intensity, it is possible to determine whether the sample contains a target by comparing it with the curve pattern of the Cp value or Ct value and the fluorescence intensity of the internal control and the negative control.

The above method is an exemplary method for confirming the amplification result, and the method for confirming the amplification result is not limited thereto and a method well known in the art can be used.

The features and advantages of the present invention are summarized as follows:

The present invention provides a composition, a kit, and a method for differential diagnosis of bovine viral diarrhea virus genotypes. When the composition, kit, and method of the present invention are used, the effect of distinguishing and detecting bovine viral diarrhea virus genotypes with high sensitivity and specificity through sequential PCR reactions is exhibited.

Figure 1 is a diagram showing the results of performing type I realtime PCR of the present invention to determine whether it is type 1 or type 2 BVDV.
Figure 2 is a diagram showing the results of performing type II real-time PCR of the present invention to determine whether it is type 1a or type 1b BVDV.
Figure 3 shows the test results of samples (1, 2, 3) that showed negative non-specific reactions when evaluated with the currently used RT-PCR kit.
Figure 4 is a diagram showing the test results of a sample (7) that showed a negative reaction when evaluated with the currently used RT-PCR kit.

Hereinafter, the present invention will be described in more detail through examples. These examples are only intended to explain the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention.

Example

Throughout this specification, "%", when used to indicate the concentration of a particular substance, unless otherwise noted, is (weight/weight) % for solid/solid, (weight/volume) % for solid/liquid, and (volume/volume) % for liquid/liquid.

Example 1: Design of a kit for detecting the bovine viral diarrhea virus of the present invention

1-1. Diagnostic Procedure

(A) Type I realtime PCR: Differential detection of bovine viral diarrhea virus (BVDV) types 1 and 2 in blood samples;

(B) Type II real-time PCR: Differential diagnosis of type 1a and type 1b in bovine viral diarrhea virus (BVDV) type 1 positive samples

(C) If Type 1 is positive in Type I realtime PCR and Type II realtime PCR is negative, subtype is confirmed through gene sequencing (17 genotypes have been reported for BVDV Type 1, so differentiation is required, and in Korea, 1a and 1b are predominant)

1-2. Composition of the real-time PCR kit of the present invention

The real-time PCR kit of the present invention was applied to the BVDV strains shown in Table 1 below.

Primer and probe sequences of the kit of the present invention division Primer and Probe Sequences SEQ ID NO: Type I real-time one-step RT-PCR K110F CTAGCCATGCCCTTAGTAGGA 1 K111R AACCA Y TGACGACT N CCCTGTACTC 2 K112P FAM-ACGAACTCACCACTG Y TGCTACCC-BHQ1 3 K113P Cy5-GAACTCACTGCTACCGCTAGTCCC-BHQ2 4 Type II one-step RT-PCR K079F CTGAGTACAGGGYAGTCGT 5 K049R TGGGCRTGCCCTCGTCCAC 6 K080P FAM-TCGACRCCTTGGMATAAAGGTCTCG-BHQ1 7 K081P FAM-TCGACGCCTTGACGTAAAGGTCTCG-BHQ1 8 K082P HEX-TCGACGCTTTGGAGRACAAGCCTCG-BHQ1 9 K083P HEX-TCGACGCTTCGTGTGACAAGCCTCG-BHQ1 10

Target location information of the primer and probe sequences of the kit of the present invention division Primer and Probe Sequences mer LOD site K110F CTAGCCATGCCCTTAGTAGGA 21 102bp 102-122* K111R AACCA Y TGACGACT N CCCTGTACTC 25 179-203* K112P FAM-ACGAACTCACCACTG Y TGCTACCC-BHQ1 24 138-161* K113P Cy5-GAACTCACTGCTACCGCTAGTCCC-BHQ2 24 134-157**

*AJ133738 (NADL standard)**NC_039237 (890 standard)

Target location information of the primer and probe sequences of the kit of the present invention division Primer and Probe Sequences mer LOD site K079F CTGAGTACAGGGYAGTCGT 19 79bp 177-195* K049R TGGGCRTGCCCTCGTCCAC 19 237-255* K080P FAM-TCGACRCCTTGGMATAAAGGTCTCG-BHQ1 25 203-227* K081P FAM-TCGACGCCTTGACGTAAAGGTCTCG-BHQ1 25 203-227* K082P HEX-TCGACGCTTTGGAGRACAAGCCTCG-BHQ1 25 202-226** K083P HEX-TCGACGCTTCGTGTGACAAGCCTCG-BHQ1 25 202-226**

*AJ133738 (NADL standard)**M96687 (Osloss standard)

1-3. Reaction conditions of the real-time PCR kit of the present invention

Reaction conditions of the real-time PCR kit of the present invention step temperature (degrees) hour cycle reverse transcription (RT) 50 30 minutes 1 initial denature 95 15 minutes 1 denaturation 95 10 seconds 40 annealing % extension 60 60 seconds

The reaction of the real-time PCR kit for detecting BVDV of the present invention was performed according to the temperature and time conditions as shown in Table 5 above.

Example 2: Evaluation of the efficacy of the differential diagnosis kit for bovine viral diarrhea virus of the present invention

2-1. Sensitivity

Type I real-time PCR of the present invention having the composition shown in Table 2 was performed on the virus strains shown in Table 1 above, and the results are shown in Figure 1.

As shown in Figure 1, it was confirmed that positive samples of type 1 or type 2 could be detected up to 1 copy/μl for all virus strains.

In addition, the type II real-time PCR of the present invention having the composition shown in Table 2 was performed on a positive sample identified as type 1 BVDV, and the results are shown in Fig. 2.

As shown in Figure 2, it was confirmed that for all virus strains identified as type 1 BVDV, it was possible to detect positive samples of type 1a or type 1b up to 1 copy/μl.

2-2. Specificity

In order to confirm the specificity of the kit of the present invention, the inventors confirmed cross-reaction using the nucleic acids of microorganisms shown in Table 6 below, which they had in their laboratory. As a result, it was confirmed that no positive reaction occurred in any other genomes except for the positive control group.

turn microorganism Genome registration number EHV-1
(FAM) EHV-4
(VIC) IC
(Cy5) 1 Bacillus cereus KCTC 1013 - - + 2 Bacillus subtilis KCTC 2213 - - + 3 Bacillus thuringiensis KCTC 1108 - - + 4 E. coli KCCM 11234 - - + 5 Listeria monocytogenes ATCC 15313 - - + 6 Salmonella enterica subsp. arizonae KCTC 12398 - - + 7 Salmonella bongori KCTC 12397 - - + 8 Salmonella enterica subsp. enterica KTCC 9120 - - + 9 Salmonella enterica subsp. enterica ATCC 43971 - - + 10 Salmonella enteritidis ATCC 13076 - - + 11 Salmonella enterica subsp. Houtenae ATCC 43974 - - + 12 Salmonella typhimurium ATCC 13311 - - + 13 Shigellasonnei ATCC 9290 - - + 14 Staphylococcus aureus ATCC 13565 - - + 15 Yersinia enterocolitica KCCM 41657 - - + 16 Vibrio vulnificus KCTC 2980 - - + 17 Vibrio vunificus KCTC 2982 - - + 18 Salmonella enteritidis KCTC 12400 - - + 19 Listeria monocytogenes ATCC 19115 - - + 20 E.coli O157:H7 ATCC 43888 - - + 21 E.coli O157:H7 ATCC 43894 - - + 22 E.coli O157:H7 ATCC 43890 - - + 23 E. coli O78:K80(B):H12 ST ATCC 43896 - - + 24 E.coli O91:H21 ATCC 51434 - - + 25 E.coli O25:K98:HNM LT ATCC 43886 - - + 26 E.coli O157:H7 ATCC 43889 - - + 27 E.coli O78:H11 LT, ST ATCC 35401 - - + 28 E.coli O157:H7 ATCC 43895 - - + 29 Vibrio parahaemolyticus ATCC 17802 - - + 30 Staphylococcus aureus ATCC 27664 - - + 31 Staphylococcus aureus NCCP 11472 - - + 32 Staphylococcus aureus ATCC 27664 - - + 33 Staphylococcus aureus ATCC 13565 - - + 34 Staphylococcus aureus ATCC 14458 - - + 35 Staphylococcus aureus ATCC 19095 - - + 36 Staphylococcus aureus ATCC 23235 - - + 37 Staphylococcus aureus ATCC 10832 - - + 38 Staphylococcus aureus ATCC 25923 - - + 39 Staphylococcus aureus ATCC 6538 - - + 40 Vibrio vulnificus ATCC 27562 - - + 41 Equus caballus meat - - - + 42 Gallus gallus meat - - - + 43 Sus scrofa meat - - - + 44 Bos taurus meat - - - + 45 Human papilloma virus 16 - - - + 46 Norovirus (GⅠ) - - - + 47 Rotavirus (01-029) - - - + 48 Adenovirus - - - + 49 No template control - - - + 50 Mumps virus - - - + 51 Influenza virus H3N2 - - - + 52 PRRSV/PCV2 - - - + 53 Parainfluenza virus 1 - - - + 54 Parainfluenza virus 2 - - - + 55 Parainfluenza virus 3 - - - + 56 Respiratory syncytial virus A - - - + 57 Respiratory syncytial virus B - - - + 58 Coronavirus OC43 - - - + 59 Coronavirus 229E - - - + 60 Coronavirus NL63 - - - + 61 Akabane virus - - - + 62 Aino virus - - - + 63 Chuzan virus - - - + 64 Ibaraki virus - - - + 65 bovine ephemeral fever virus - - - + 66 bovine herpes virus 1 - - - + 67 Positive control - + + +

2-3. Validity evaluation using outdoor samples

The present inventors detected the genotypes of BVDV using the kit of the present invention for BVDV 1a isolates (5 strains), BVDV 1b isolates (5 strains), BVDV 2a isolates (5 strains), BVDV 1o isolates (1 strain), and BVDV 1n (1 strain). As a result, the BVDV genotypes were accurately detected for all field samples, and the sensitivity evaluation results showed that detection was possible up to 1xTCID ₅₀ /ml for all. The results are shown in Table 7.

division Type I real-time one-step RT-PCR Type II real-time one-step RT-PCR BVDV type 1 BVDV type 2 BVDV 1a BVDV 1b BVDV (1a) Type 1(+) - 1a(+) - BVDV (1a) Type 1(+) - 1a(+) - BVDV (1a) Type 1(+) - 1a(+) - BVDV (1a) Type 1(+) - 1a(+) - BVDV (1a) Type 1(+) - 1a(+) - BVDV (1b) Type 1(+) - - 1b(+) BVDV (1b) Type 1(+) - - 1b(+) BVDV (1b) Type 1(+) - - 1b(+) BVDV (1b) Type 1(+) - - 1b(+) BVDV (1b) Type 1(+) - - 1b(+) BVDV (2a) - Type 2(+) - - BVDV (2a) - Type 2(+) - - BVDV (2a) - Type 2(+) - - BVDV (2a) - Type 2(+) - - BVDV (2a) - Type 2(+) - - BVDV (1o) Type 1(+) - - - BVDV (1n) Type 1(+) - - -

2-4. Comparative evaluation with RT-PCR kits currently approved and used for livestock quarantine

The RT-PCR kit currently approved and used for livestock quarantine is the BVDV common PCR (cat. LiliF ^TM IP12040), which is a kit that does not allow genotypic differentiation.

As a result of evaluating 20 BVDV 1a, 1b, and 2a positive tissue samples, all were confirmed to be positive with the type I and type II real-time PCR kits of the present invention.

In addition, as a result of evaluating 60 negative samples using the type I, type II real-time PCR kit of the present invention, 42 samples were confirmed to be negative and 18 samples were confirmed to be positive.

Meanwhile, among the samples that were evaluated as negative using the currently used RT-PCR kit and confirmed as weakly positive with a Ct value of 33 or more and 38 or less using the newly developed kit of the present invention, all were confirmed to be BVDV positive as a result of base sequence analysis.

In addition, among the samples that were evaluated as negative using the currently used RT-PCR kit and had a strong non-specific reaction, 15 samples were evaluated using the newly developed kit of the present invention and all were confirmed to be negative without any non-specific reaction.

From the above results, it was confirmed that the novel development kit of the present invention has higher sensitivity and specificity than existing RT-PCR kits.

Figure 3 shows the test results of samples (1, 2, 3) that showed negative non-specific reactions when evaluated with the currently used RT-PCR kit.

In Fig. 3, a sample that showed a negative non-specific reaction when evaluated with the currently used RT-PCR kit was confirmed to be negative when evaluated with the new development kit of the present invention.

Figure 4 is a diagram showing the test results of a sample (7) that showed a negative reaction when evaluated with the currently used RT-PCR kit.

In Fig. 4, a sample that showed a negative reaction when evaluated with the currently used RT-PCR kit showed a positive reaction when evaluated with the new development kit of the present invention.

Evaluation of the development diagnostic method in positive test samples Serial number division Test substance Type I real-time one-step RT-PCR Type II real-time one-step RT-PCR BVDV type 1 BVDV type 2 BVDV 1a BVDV 1b 1 BVDV 1a
positivity Whole blood 1 + - + - 2 Whole blood 2 + - + - 3 Whole blood 3 + - + - 4 Whole blood 4 + - + - 5 Whole blood 5 + - + - 6 Aborted fetus 1 + - + - 7 Abortion Fetus 2 + - + - 8 Abortion Fetus 3 + - + - 9 Aborted fetus 4 + - + - 10 Abortion Fetus 5 + - + - 11 lung1 + - + - 12 lung 2 + - + - 13 lung 3 + - + - 14 lung 4 + - + - 15 lung 5 + - + - 16 Feces 1 + - + - 17 Feces 2 + - + - 18 Feces 3 + - + - 19 Feces 4 + - + - 20 Feces 5 + - + - 21 BVDV 1b
positivity Whole blood 6 + - - + 22 Whole blood 7 + - - + 23 Whole blood 8 + - - + 24 Whole blood 9 + - - + 25 Whole blood 10 + - - + 26 Aborted fetus 6 + - - + 27 Abortion Fetus 7 + - - + 28 Abortion fetus 8 + - - + 29 Aborted fetus 9 + - - + 30 Abortion Fetus 10 + - - + 31 lung 6 + - - + 32 lung7 + - - + 33 lung8 + - - + 34 lung9 + - - + 35 Lung 10 + - - + 36 Feces 6 + - - + 37 Feces 7 + - - + 38 Feces 8 + - - + 39 Feces 9 + - - + 40 Feces 10 + - - + 41 BVDV 2a
positivity Whole blood 11 - + - - 42 Whole blood 12 - + - - 43 Whole blood 13 - + - - 44 Whole blood 14 - + - - 45 Whole blood 15 - + - - 46 Miscarriage fetus 11 - + - - 47 Miscarriage fetus 12 - + - - 48 Miscarriage fetus 13 - + - - 49 Miscarriage fetus 14 - + - - 50 Miscarriage fetus 15 - + - - 51 Lung 11 - + - - 52 Lung 12 - + - - 53 Lung 13 - + - - 54 Lung 14 - + - - 55 Lung 15 - + - - 56 Feces 11 - + - - 57 Feces 12 - + - - 18 Feces 13 - + - - 59 Feces 14 - + - - 60 Feces 15 - + - -

Evaluation of developed diagnostic methods from voice test samples Serial number Test substance Type I real-time one-step RT-PCR Type II real-time one-step RT-PCR BVDV type 1 BVDV type 2 BVDV 1a BVDV 1b 1 Whole blood 1 +
(36.3) - +
(37.12) - 2 Whole blood 2 - - - - 3 Whole blood 3 - - - - 4 Whole blood 4 - - - - 5 Whole blood 5 - - - - 6 Aborted fetus 1 +
(34.6) - +
(35.2) - 7 Aborted fetus 2 +(36.9) - +
(37.4) - 8 Abortion Fetus 3 - - - - 9 Aborted fetus 4 - - - - 10 Abortion Fetus 5 - - - - 11 lung1 - - - - 12 lung 2 - - - - 13 lung 3 - - - - 14 lung 4 - - - - 15 lung 5 +
(35.6) - +
(36.4) - 16 Feces 1 +(36.8 - +
(37.4) - 17 Feces 2 - - - - 18 Feces 3 - - - - 19 Feces 4 - +
(33.8) - - 20 Feces 5 - +(37.1) - - 21 Whole blood 6 - +(38.5) - - 22 Whole blood 7 - +(34.6) - - 23 Whole blood 8 - - - - 24 Whole blood 9 - - - - 25 Whole blood 10 - - - - 26 Aborted fetus 6 - +
(33.8) - - 27 Abortion Fetus 7 - +(358) - - 28 Abortion fetus 8 - - - - 29 Aborted fetus 9 - - - - 30 Abortion Fetus 10 - - - - 31 lung 6 - +(33.4) - - 32 lung7 - - - - 33 lung8 - - - - 34 lung9 - - - - 35 Lung 10 - - - - 36 Feces 6 - - - - 37 Feces 7 - - - - 38 Feces 8 - - - - 39 Feces 9 - - - - 40 Feces 10 - - - - 41 Whole blood 11 +(36.2) - - + 42 Whole blood 12 + - - + 43 Whole blood 13 - - - - 44 Whole blood 14 - - - - 45 Whole blood 15 - - - - 46 Miscarriage fetus 11 - +
(34.6) - - 47 Miscarriage fetus 12 - +
(35.3) - - 48 Miscarriage fetus 13 - - - - 49 Miscarriage fetus 14 - - - - 50 Miscarriage fetus 15 - - - - 51 Lung 11 - - - - 52 Lung 12 - - - - 53 Lung 13 - - - - 54 Lung 14 - - - - 55 Lung 15 - - - - 56 Feces 11 - - - - 57 Feces 12 - - - - 18 Feces 13 +(33.6) - - +
(34.9) 59 Feces 14 +(35.2) - - +
(35.9) 60 Feces 15 +(37.4) - - +
(38.1)

<110> REPUBLIC OF KOREA(Animal and Plant Quarantine Agency) <120> Kit and method for differential diagnosis of Bovine viral diarrhea virus subgenotype <130> PN210436 <160> 10 <170> KoPatentIn 3.0 <210> 1 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> K110F <400> 1 ctagccatgc ccttagtagg a 21 <210> 2 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> K111R <400> 2 aaccaytgac gactnccctg tactc 25 <210> 3 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> K112P <400> 3 acgaactcac cactgytgct accc 24 <210> 4 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> K113P <400> 4 gaactcactg ctaccgctag tccc 24 <210> 5 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> K079F <400> 5 ctgagtacag ggyagtcgt 19 <210> 6 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> K049R <400> 6 tgggcrtgcc ctcgtccac 19 <210> 7 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> K080P <400> 7 tcgacrcctt ggmataaaagg tctcg 25 <210> 8 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> K081P <400> 8 tcgacgcctt gacgtaaagg tctcg 25 <210> 9 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> K082P <400> 9 tcgacgcttt ggagracaag cctcg 25 <210> 10 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> K083P <400> 10 tcgacgcttc gtgtgacaag cctcg 25

Claims (17)

Composition for differential diagnosis of bovine viral diarrhea virus genotypes, comprising:
(i) a primer pair for detecting bovine viral diarrhea virus (BVDV) genotype 1 or 2 consisting of the nucleotide sequences of sequence numbers 1 and 2, and a probe for differential diagnosis of BVDV type 1 consisting of the nucleotide sequence of sequence number 3; and a probe for differential diagnosis of BVDV type 2 consisting of the nucleotide sequence of sequence number 4; and
(ii) a primer pair for detecting BVDV genotype 1a or 1b consisting of the nucleotide sequences of SEQ ID NOs: 5 and 6, and a probe for differential diagnosis of BVDV type 1a consisting of the nucleotide sequence of SEQ ID NO: 7; a probe for differential diagnosis of BVDV type 1a consisting of the nucleotide sequence of SEQ ID NO: 8; a probe for differential diagnosis of BVDV type 1b consisting of the nucleotide sequence of SEQ ID NO: 9; and a probe for differential diagnosis of BVDV type 1b consisting of the nucleotide sequence of SEQ ID NO: 10.
delete A composition in claim 1, wherein the 5'-terminal of the nucleotide sequence of sequence number 3 and sequence number 4 is labeled with one different fluorophore selected from the group consisting of FAM, TET, HEX, JOE, ROX, TMR, Cy5, TxR, and Cal Red 610, and the 3'-terminal is modified with a quencher of BHQ-1, BHQ-2, or BHQ-3.
delete delete A composition in claim 1, wherein the 5'-terminal of the probe consisting of any one of the nucleotide sequences of SEQ ID NOs: 7 to 10 is labeled with a fluorophore selected from the group consisting of FAM, TET, HEX, JOE, ROX, TMR, Cy5, TxR, and Cal Red 610, and the 3'-terminal is modified with a quencher of BHQ-1, BHQ-2, or BHQ-3.
A composition in claim 6, wherein the probe comprising the nucleotide sequence of sequence number 7 and the probe comprising the nucleotide sequence of sequence number 8 are labeled with at least one fluorescent substance that is the same as or different from each other.
A composition in claim 6, wherein the probe comprising the nucleotide sequence of sequence number 9 and the probe comprising the nucleotide sequence of sequence number 10 are labeled with at least one fluorescent substance that is the same as or different from each other.
A kit for differential diagnosis of bovine viral diarrhea virus genotypes, comprising a composition according to any one of claims 1, 3, and 6 to 8.
In claim 9, the kit is a kit for differential diagnosis of bovine viral diarrhea virus genotypes, which is performed by gene amplification.
A kit for differential diagnosis of bovine viral diarrhea virus genotypes, wherein the gene amplification in claim 10 is performed by PCR (polymerase chain reaction).
A kit for differential diagnosis of bovine viral diarrhea virus genotypes, wherein the gene amplification in claim 10 is performed by real-time PCR.
Differential diagnosis method for bovine viral diarrhea virus genotypes, including the following steps:
(a) a step of preparing nucleic acid from a specimen collected from a cow or the environment;
(b) a step of amplifying the target nucleotide sequence in the test sample using the composition of any one of claims 1, 3, and 6 to 8; and
(c) A step of confirming the above amplification results.
A method in claim 13, wherein the amplification is performed by PCR (polymerase chain reaction).
A method according to claim 13, wherein the amplification is performed by real-time PCR.
A method in claim 15, wherein the real-time PCR is performed using a TaqMan probe method.
In claim 16, a method wherein the probe is bound to a label, and the confirmation in step (c) is performed by detecting a signal generated from the probe.