CN113136455A - Multiplex fluorescence quantitative PCR method and kit for detecting BVDV, BCoV, BRV and IBRV - Google Patents
Multiplex fluorescence quantitative PCR method and kit for detecting BVDV, BCoV, BRV and IBRV Download PDFInfo
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Abstract
The invention discloses a multiple fluorescence quantitative PCR method and a kit for detecting BVDV, BCoV, BRV and IBRV, belonging to the technical field of veterinary biological diagnosis. The nucleotide sequence of the primer group provided by the invention is shown in SEQ ID NO.1-SEQ ID NO.8, the simultaneous and quantitative detection of bovine viral diarrhea virus, bovine coronavirus, bovine rotavirus and bovine infectious rhinotracheitis virus is realized, and the detection method has high sensitivity and good specificity and is suitable for the detection of clinical samples.
Description
Technical Field
The invention belongs to the technical field of veterinary biological diagnosis, and particularly relates to a multiple fluorescence quantitative PCR method and a kit for detecting BVDV, BCoV, BRV and IBRV.
Background
Newborn calf diarrhea is a common disease in clinic, causes serious harm to the health of calves, and is one of important reasons for causing economic loss of dairy cow industry. Pathogens causing calf diarrhea are many, including viruses, bacteria and parasites, among which the viruses are mainly Bovine Viral Diarrhea Virus (BVDV), Bovine Coronavirus (BCoV), Bovine Rotavirus (BRV), and Infectious Bovine Rhinotracheitis Virus (IBRV), and the like, and are worldwide prevalent. Bovine viral diarrhea virus belongs to the genus pestivirus of the flaviviridae family, is a single-stranded positive-strand RNA virus, cattle infected with the virus mainly show diarrhea, mucosal cyanosis, digestive tract mucosal erosion, immune tolerance, persistent infection and the like, BVDV naturally infected objects are cows and yellow cattle, the virus causes a contact infectious disease-Bovine viral diarrhea/mucosal disease (Bovine viral diarrheea/mucosae disease, BVD/MD, serious harm; Bovine coronavirus belongs to the genus coronavirus of the family coronavirus, is an unsegmented single-stranded positive-strand RNA virus, is an important pathogen causing diarrhea and respiratory tract infection of calves, calf infected with BCoV mainly shows diarrhea, can also cause winter diarrhea of adult calves, respiratory tract infection causes Bovine pneumonia and the like; Bovine rotavirus belongs to the family reoviridae, the genus rotavirus, is an important pathogen causing diarrhea of calves, cattle are diseased watery excrement like, and often cause secondary infections. Infectious bovine rhinotracheitis virus can cause an acute, thermal and contact infectious disease of cattle, cattle infected with the virus are mainly manifested as respiratory symptoms and are one of important pathogens causing enteritis type diarrhea of cattle, sick cattle can sometimes have bloody diarrhea, and newborn calves can be manifested as systemic diseases.
With the continuous development of scientific technology in China, the living standard of people is continuously improved, the breeding industry is also continuously expanded, calf diarrhea diseases caused by four viruses of BVDV, BCoV, BRV and IBRV seriously affect the health of cows, bring great influence to the cow industry in China and cause huge economic loss. No effective treatment medicine or preventive measure for the diseases caused by the four viruses exists, and the European countries adopt the method of detecting and eliminating the diseased cattle as soon as possible to eradicate the diseases. The existing detection method mainly depends on virus separation and identification, enzyme-linked immunosorbent assay (ELISA), neutralization assay, indirect Immunofluorescence (IFA) technology, common PCR technology, real-time fluorescent quantitative PCR detection technology and the like. However, these test methods have the disadvantages of long period, low detection amount, high cost, complex operation, poor specificity and the like, so that a multiplex fluorescence quantitative PCR detection method which is rapid, sensitive and stable and can detect four viruses, i.e., BVDV, BCoV, BRV and IBRV, is urgently needed to be established, and a rapid and effective detection technology is provided for clinical detection.
Disclosure of Invention
One of the purposes of the invention is to provide a multiplex SYBR Green I fluorescent quantitative PCR primer group for detecting BVDV, BCoV, BRV and IBRV.
The invention also aims to provide a multiplex SYBR Green I fluorescent quantitative PCR method for detecting BVDV, BCoV, BRV and IBRV.
The invention also aims to provide a kit for detecting BVDV, BCoV, BRV and IBRV.
The technical purpose of the invention is realized by the following technical scheme:
a multiple fluorescent quantitative PCR primer group for detecting BVDV, BCoV, BRV and IBRV comprises 4 pairs of primers, and the nucleotide sequences of the primers are shown as SEQ ID NO.1-SEQ ID NO. 8:
BVDV-F：5'-ACAGGGCAGTCGTCAGTG-3'(SEQ ID NO.1)
BVDV-R：5'-TTCGTAGCAGTCGGTTGG-3'(SEQ ID NO.2)
BCoV-F：5'-AGTTTGAATTTGCAGAGGGACA-3'(SEQ ID NO.3)
BCoV-R：5'-GGTTGCCATCGGCTGTTT-3'(SEQ ID NO.4)
BRV-F:5'-AGATAATGTATGTATGGACGAG-3'(SEQ ID NO.5)
BRV-R:5'-GACGCTGAATAAGGGAAA-3'(SEQ ID NO.6)
IBRV-F：5'-GCAAGGTGGTGGCCTTCGAC-3'(SEQ ID NO.7)
IBRV-R：5'-GAGGTGCCCGTGCGGTAGAG-3'(SEQ ID NO.8)
wherein the BVDV-F/R, BCoV-F/R, BRV-F/R and the IBRV-F/R can be specifically combined with a target gene respectively, and specifically amplify the following genes: the 5' UTR gene of BVDV, the N gene of BCoV, the VP6 gene of BRV and the gB gene of IBRV.
Amplifying a primer pair BVDV-F/R to obtain a target gene fragment with the size of 137 bp; the target gene fragment amplified by the primer pair BCoV-F/R is 117 bp; amplifying a BRV-F/R primer pair to obtain a target gene fragment with the size of 213 bp; the target gene fragment obtained by amplification of a primer pair IBRV-F/R has the size of 151 bp.
Meanwhile, the application of the primer group in detecting BVDV, BCoV, BRV and IBDV or in preparing a BVDV, BCoV, BRV and IBDV detection kit is also in the protection scope of the invention.
A multiple fluorescent quantitative PCR method for detecting BVDV, BCoV, BRV and IBRV comprises the following steps:
(1) extracting sample nucleic acid and carrying out reverse transcription;
(2) respectively carrying out multiple fluorescent quantitative PCR amplification reactions on the mixture obtained by reverse transcription in the step (1) and positive plasmids of 4 viruses by using primers SEQ ID NO.1-SEQ ID NO.8 to obtain a PCR amplification curve;
(3) and (3) analyzing results according to the amplification curve obtained in the step (2).
Preferably, the sample in step (1) is feces.
Preferably, said step(2) The total concentration of each component in the amplification system of the medium-multiplex fluorescent quantitative PCR is as follows:
green qPCR Supermix 12.5. mu.L, final concentration of each upstream and downstream primer 0.2uM/uL, template/plasmid standard 2. mu.L, using ddH2O makes up 25 uL.
Preferably, the amplification procedure of the multiplex quantitative fluorescence PCR in the step (2) is as follows: 300s at 95 ℃, 10s at 57 ℃ and 25s at 72 ℃ for 40 cycles.
In addition, the application of the multiple fluorescent quantitative PCR method for BVDV, BCoV, BRV and IBRV in the preparation of the detection kit is also within the protection scope of the invention.
Meanwhile, the invention also provides a kit for detecting BVDV, BCoV, BRV and IBRV, wherein the kit comprises a primer group shown by SEQ ID NO.1-SEQ ID NO. 8.
Preferably, the kit further comprises positive plasmids of BVDV, BCoV, BRV and IBRV, and a fluorescence quantitative PCR standard curve can be constructed by the positive plasmids.
Preferably, reagents required for fluorescent quantitative PCR, such as ExTaq buffer, dNTPs, MgCl, are also included2And the like.
Meanwhile, the application of the kit in detection of BVDV, BCoV, BRV and IBRV is also within the protection scope of the invention.
As a preferred embodiment, the method for detecting BVDV, BCoV, BRV and IBRV by using the kit comprises the following steps:
(1) extracting sample nucleic acid and carrying out reverse transcription;
(2) performing multiplex fluorescence quantitative PCR amplification reaction on the mixture obtained by reverse transcription in the step (1) and the positive plasmids of the 4 viruses respectively by using primers SEQ ID NO.1-SEQ ID NO.8 to obtain a PCR amplification curve;
(3) and (3) analyzing results according to the amplification curve obtained in the step (2).
Preferably, the total concentration of each component in the amplification system of the multiplex fluorescence quantitative PCR in the step (2) is as follows:
green qPCR Supermix 12.5. mu.L, final concentration of each upstream and downstream primer 0.2uM/uL, template/plasmid standard 2. mu.L, using ddH2O makes up 25 uL.
Preferably, the amplification procedure of the multiplex quantitative fluorescence PCR in the step (2) is as follows: 300s at 95 ℃, 10s at 57 ℃ and 25s at 72 ℃ for 40 cycles.
Compared with the prior art, the invention has the following beneficial effects:
the primer group provided by the invention realizes simultaneous and quantitative detection of bovine viral diarrhea virus, bovine coronavirus, bovine rotavirus and bovine infectious rhinotracheitis virus, and the detection method has high sensitivity and good specificity and is suitable for detection of clinical samples.
Drawings
FIG. 1 is a graph showing the results of PCR amplification of the BVDV recombinant plasmid, BCoV recombinant plasmid, BRV recombinant plasmid and IBRV recombinant plasmid in example 1.
FIG. 2a is a graph showing the BVDV multiplex fluorescent quantitative PCR amplification in example 3.
FIG. 2b is a graph of the BCoV multiplex fluorescent quantitative PCR amplification in example 3.
FIG. 2c is a graph of the multiplex quantitative PCR amplification of BRV in example 3.
FIG. 2d is a graph of the multiplex quantitative PCR amplification of IBRV in example 3.
FIG. 3a is a standard curve diagram of BVDV multiplex quantitative PCR in example 3.
FIG. 3b is a standard curve of BCoV multiplex quantitative PCR in example 3.
FIG. 3c is a graph of the standard BRV multiplex quantitative PCR in example 3.
FIG. 3d is a graph of the standard IBRV multiplex quantitative PCR in example 3.
FIG. 4 is a graph showing the dissolution profiles of the four primers in multiplex quantitative PCR in example 3.
FIG. 5 is a graph showing the results of the specificity of the multiplex quantitative PCR detection method in comparative example 1.
FIG. 6 is a graph showing the results of fluorescent quantitative PCR amplification of the target fragment with 4 pairs of primers in example 4.
FIG. 7a is a graph showing the results of sensitivity measurement of BVDV fluorescent quantitative PCR in example 5.
FIG. 7b is a graph showing the results of sensitivity detection by fluorescent quantitative PCR of BRV in example 5.
FIG. 7c is a graph showing the results of sensitivity detection of BCoV fluorescent quantitative PCR in example 5.
FIG. 7d is a graph showing the results of sensitivity detection by IBRV fluorescent quantitative PCR in example 5.
Detailed Description
Unless otherwise indicated, the reagents and materials used in the following examples are commercially available and the dye used in the fluorescent PCR is SYRBGreen I.
Example 1
Designing a primer:
designing a fluorescent quantitative specific amplification primer group according to BVDV, BCoV, BRV and IBRV genomes, wherein the specific sequences are as follows:
BVDV-F：5'-ACAGGGCAGTCGTCAGTG-3'(SEQ ID NO.1)
BVDV-R：5'-TTCGTAGCAGTCGGTTGG-3'(SEQ ID NO.2)
BCoV-F：5'-AGTTTGAATTTGCAGAGGGACA-3'(SEQ ID NO.3)
BCoV-R：5'-GGTTGCCATCGGCTGTTT-3'(SEQ ID NO.4)
BRV-F:5'-AGATAATGTATGTATGGACGAG-3'(SEQ ID NO.5)
BRV-R:5'-GACGCTGAATAAGGGAAA-3'(SEQ ID NO.6)
IBRV-F：5'-GCAAGGTGGTGGCCTTCGAC-3'(SEQ ID NO.7)
IBRV-R：5'-GAGGTGCCCGTGCGGTAGAG-3'(SEQ ID NO.8)。
example 2
Preparation of positive plasmids of BVDV, BCoV, BRV and IBRV, comprising the following steps:
(1) extracting sample nucleic acid and carrying out reverse transcription;
(2) performing common PCR amplification by using the mixture obtained by reverse transcription in the step (1) as a template and utilizing primer groups of SEQ ID NO.1-SEQ ID NO.8 respectively;
the total PCR reaction was 25 μ L: 1. mu.L of cDNA (DNA) template,the final concentration of the upstream and downstream primers was 0.2uM/uL, 2 × Phanta Max Master Mix 12.5 μ L, ddH2O to complement 25 μ L;
the general PCR target fragment amplification reaction program of BVDV and BCoV is as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 56 ℃ for 30s, and extension at 72 ℃ for 15s, for 35 cycles, and final extension at 72 ℃ for 7 min;
the BRVPCR reaction procedure was: pre-denaturation at 95 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 15s for 35 cycles, and final extension at 72 ℃ for 7 min;
the IBRVPCR reaction program is as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 57 ℃ for 30s, and elongation at 72 ℃ for 15s for 35 cycles, and final elongation at 72 ℃ for 7 min.
(3) Taking 5 mu L of PCR product obtained by amplification to carry out agarose gel electrophoresis detection, wherein the result is shown in figure 1, and M is DL 1000 Marker; 1 is BVDV 5' UTR gene; 2 is BCoV N gene; 3 is BRV VP6 gene; 4 is IBRV gB gene; and 5 is a negative control. An amplified band with the size of 137bp appears in the electrophoretogram, which indicates that the sample BVDV is detected to be positive; an amplified band with the size of 117bp appears in the electrophoretogram, which indicates that the BCoV detection in the sample is positive; an amplified band with the size of 213bp appears in the electrophoretogram, which indicates that the BRV detection in the sample is positive; an amplified band with the size of 151bp appears in the electrophoretogram, which indicates that the IBRV detection result in the sample is positive.
(4) And (2) performing gel recovery and purification on specific target bands obtained by amplifying the four viruses, respectively cloning target fragments obtained by gel recovery and purification to a pUC-57 vector, transforming the target fragments to DH5 alpha competent cells, culturing a transformation product, extracting plasmids, detecting positive clones by PCR (polymerase chain reaction) and sending the positive clones to Jilin province American biotechnology and technology Limited company for sequencing, wherein sequencing results show that amplified fragments with correct sequences are obtained, and the concentration of the plasmids is determined by using Nanodrop 2000, and the copy number of the plasmids is calculated and used as a plasmid standard product.
Example 3
A method for detecting BVDV, BCoV, BRV and IBRV by real-time multiplex fluorescence quantitative PCR comprises the following steps:
weighting pUC-57-BVDV recombinant plasmid, pUC-57-BCoV recombinant plasmid, pCU-57-BRV recombinant plasmid and pUC-57-IBRVThe plasmid was diluted 10-fold gradient to 10-fold using sterile triple-distilled water 13 replicates were made for each concentration. The PCR reaction system is as follows:
green qPCR Supermix 12.5. mu.L, final concentration of each upstream and downstream primer 0.2uM/uL, template/plasmid standard 2. mu.L, using ddH2The amount of O to be added to 25uL was adjusted to 40 cycles of 95 ℃ 300s, 95 ℃ 10s, 57 ℃ 10s, and 72 ℃ 25s, and fluorescence signals were collected after each cycle was completed. Performing multiplex fluorescence quantitative PCR amplification, taking plasmids with different concentration gradients as templates, and establishing four standard curves by taking the logarithm value of the standard substance concentration as an X axis and the Cq value as a Y axis after the amplification is finished. FIGS. 2a-2d are the multiplex quantitative PCR amplification curves for BVDV, BCoV, BRV and IBRV; FIGS. 3a-3d are four standard curves for multiplex quantitative PCR of BVDV, BCoV, BRV and IBRV; FIG. 4 shows the four primer melting curves of multiplex quantitative PCR.
Comparative example 1
The experimental procedure was as in example 4, except that the primer sets used were as follows:
BVDV-2F：5'-AGTCGTCAGTGGTTCGACGCCTTGG-3'(SEQ ID NO.9)
BVDV-2R：5'-CCTATCAGGCTGTATTCGTAA-3'(SEQ ID NO.10)
BCoV-2F：5'-GTACCCTACTATTCTTGGTTCTC-3'(SEQ ID NO.11)
BCoV-2R：5'-TCGGTGCCATACTGGTCTTT-3'(SEQ ID NO.12)
BRV-2F：5'–GTAGATAATGTATGTATGGACGAG-3'(SEQ ID NO.13)
BRV-2R：5'-ATGACGCTGAATAAGGGAAA-3'(SEQ ID NO.14)
IBRV-2F：5'–ACCTGGTGGACAAGAAGTGGCG-3'(SEQ ID NO.15)
IBRV-2R：5'–TGCGGTAGAGCCCCGCCGAGCCCAG-3'(SEQ ID NO.16)
the four pairs of primers are used for carrying out fluorescent quantitative PCR amplification on the target fragment, the result is shown in FIG. 5, and 4 pairs of primers used in the comparative example cannot uniformly amplify a fluorescent curve, which indicates that the amplification effect of the primer group is poor.
Example 4
This example carries out the specificity test of SEQ ID NO.1 to SEQ ID NO.8, with the following steps:
the extracted DNA of bovine parvovirus, bovine cryptosporidium, bovine coccidia and bovine infectious rhinotracheitis virus (IBRV) and the cDNA of reverse transcription completed Bovine Rotavirus (BRV), bovine coronavirus (BCoV) and BVDV are taken as templates, a negative control is set simultaneously, the primers are used for amplifying SEQ ID NO.1-SEQ ID NO.8 and the established multiplex fluorescence quantitative PCR (the amplification procedure is the same as in example 3), the specificity of the method is detected, and the result is shown in figure 6, wherein 1: an IBRV plasmid standard; 2: a BCoV plasmid standard; 3: a BRV plasmid standard; 4: a BVDV plasmid standard; 5: BPV; 6: bovine coccidia; 7: cryptosporidium bovis; 8: ddH2And O. As can be seen from FIG. 6, no fluorescence signal was amplified in any of the samples except for the presence of fluorescence signals in BVDV, BCoV, BRV and IBRV, indicating that the primer set had good specificity.
Example 5
This example carries out the sensitivity tests of SEQ ID NO.1 to SEQ ID NO.8, with the following steps:
plasmid standards for BVDV, BCoV, BRV and IBRV were diluted 10-fold gradient, where:
the BVDV plasmid dilution concentrations were: 1.41X 101copies/uL-1.41×107copies/uL；
The dilution concentration of BCoV plasmid is 1.17X 101copies/uL-1.17×107copies/uL；
The BRV plasmid dilution concentration is 1.36X 101copies/uL-1.36×107copies/uL；
The IBRV plasmid dilution concentration is 1.2X 101copies/uL-1.2×107copies/uL；
The diluted plasmid is used as a template, and the established multiplex fluorescence quantitative PCR detection method is used for amplification, and the reaction system and the reaction conditions are the same as those in the example 3. Meanwhile, the diluted plasmid standard substance is diluted by using common PCR and compared with the established multiplex fluorescence quantitative PCR detection method.
The results show that: multiple fluorescenceThe minimum sample concentration of BVDV detected by the light quantitative PCR detection method is 1.41 multiplied by 103The lowest sample concentration of copies/uL, BCoV was 1.17X 103The lowest sample concentration of copies/uL, BRV is 1.36X 103The minimum sample concentration of copies/uL, IBRV is 1.20X 103copies/uL; the minimum detection concentration of BVDV by ordinary PCR is 1.41 multiplied by 105The lowest detection concentration of copies/uL and BCoV is 1.17 multiplied by 105The lowest detection concentration of copies/uL and BRV is 1.36 multiplied by 105copies/uL, the minimum detection concentration of IBRV is 1.20 multiplied by 105copies/uL. The sensitivity of the multiplex fluorescent quantitative PCR detection method established by the test is 100 times higher than that of the common PCR method, which shows that the established multiplex fluorescent quantitative PCR detection method has better sensitivity. The results of the sensitivity test of the multiplex fluorescent quantitative PCR detection method are shown in FIGS. 7a to 7 d.
Example 6
This example carries out the reproducibility test of SEQ ID NO.1 to SEQ ID NO.8, the procedure being as follows:
respectively using BVDV1.41 × 103-1.41×105copies/uL、BCoV1.17×103-1.17×105copies/uL、BRV1.36×103-1.36×105copies/uL、IBRV1.20×103-1.20×105Taking the copies/uL3 10-fold dilution gradient diluted plasmid standard products as templates, detecting by using the established multiplex fluorescence quantitative PCR detection method, and repeating each concentration gradient for 3 times to perform an in-group repeatability test; and then 3 different times are respectively selected to carry out the intergroup repeated test on the plasmid standard substances with 3 concentration gradients. The results show that the intra-group variation coefficients are respectively 0.128-0.807% of BVDV, 0.589-1.437% of BCoV, 1.571-2.708% of BRV and 1.049-3.835% of IBRV; the coefficient of variation among groups is respectively 0.713-1.493% of BVDV, 1.046-1.736% of BCoV, 1.611-2.380% of BRV and 0.340-1.833% of IBRV, and the coefficient of variation among groups and between groups is less than 4%. The established multiplex fluorescence quantitative PCR detection method has good repeatability. The results of the reproducibility test are shown in Table 1.
Therefore, the multiplex SYRB GreenI fluorescence quantitative PCR detection method established in the test can quickly, accurately and quantitatively detect four pathogens of BVDV, BCoV, BRV and IBRV, provides an effective means for detecting calf diarrhea pathogens, and has important significance for diagnosing calf diarrhea.
TABLE 1 results of the in-and between-group repeatability tests of multiplex fluorescent quantitative PCR
Example 7
This example provides a kit for detecting BVDV, BCoV, BRV and IBRV, which comprises the primer set of example 1, positive plasmids of BVDV, BCoV, BRV and IBRV, a standard curve for fluorescence quantitative PCR constructed by the positive plasmids, and reagents required for fluorescence quantitative PCR, such as ExTaq buffer, dNTPs, MgCl2And the like.
The amplification system and the setting of amplification parameters of the kit were the same as in example 3.
The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.
Sequence listing
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<120> multiple fluorescent quantitative PCR method and kit for detecting BVDV, BCoV, BRV and IBRV
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Claims (10)
1. A multiplex SYBR Green I fluorescent quantitative PCR primer group for detecting BVDV, BCoV, BRV and IBRV is characterized by comprising 4 pairs of primers, wherein the nucleotide sequence of the primer is shown as SEQ ID NO.1-SEQ ID NO. 8.
2. Use of the primer set according to claim 1 for the detection of non-disease diagnostic purposes of BVDV, BCoV, BRV and IBRV or for the preparation of BVDV, BCoV, BRV and IBRV detection kits.
3. A multiplex SYBR Green I fluorescent quantitative PCR method for detecting non-disease diagnosis purposes of BVDV, BCoV, BRV and IBRV is characterized by comprising the following steps:
(1) extracting sample nucleic acid and carrying out reverse transcription;
(2) respectively carrying out multiple fluorescent quantitative PCR amplification reactions on the mixture obtained by reverse transcription in the step (1) and positive plasmids of 4 viruses by using primers SEQ ID NO.1-SEQ ID NO.8 to obtain a PCR amplification curve;
(3) and (3) analyzing results according to the amplification curve obtained in the step (2).
4. The method of claim 3, wherein the total concentration of each component in the amplification system of the multiplex quantitative PCR in the step (2) is as follows:
green qPCR Supermix 12.5. mu.L, final concentration of each upstream and downstream primer 0.2uM/uL, template/plasmid standard 2. mu.L, using ddH2O makes up 25 uL.
5. The method of claim 3, wherein the amplification procedure of the multiplex quantitative PCR in the step (2) is as follows: 300s at 95 ℃, 10s at 57 ℃ and 25s at 72 ℃ for 40 cycles.
6. Use of the method of any one of claims 3 to 5 in the preparation of BVDV, BCoV, BRV and IBRV detection kits.
7. A kit for detecting BVDV, BCoV, BRV and IBRV, which comprises the primer set of claim 1.
8. The kit of claim 7, further comprising BVDV, BCoV, BRV and IBRV positive plasmids.
9. The kit of claim 7, further comprising reagents required for fluorescent quantitative PCR.
10. Use of a kit according to any one of claims 7 to 9 for non-disease diagnostic purposes in the detection of BVDV, BCoV, BRV and IBRV.
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