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Multiple PCR (Polymerase Chain Reaction) detection primer set and kit for rapidly distinguishing PDCoV (Porcine deltacoronavirus) and BVDV (Bovine Viral Diarrhea Virus)
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A technology for bovine viral diarrhea and coronavirus, applied in the field of diagnosis in the field of veterinary biotechnology, can solve problems such as difficulties in clinical diagnosis
Active Publication Date: 2018-07-13
INST OF ANIMAL HUSBANDRY & VETERINARY MEDICINE HENAN ACAD OF AGRI SCI
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Problems solved by technology
Because the clinical manifestations and pathological changes of pigs infected with PDCoV and BVDV are very similar, it has caused certain difficulties in clinical diagnosis.
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Embodiment 1
[0039] 1. Primer design
[0040] According to the PDCoV and BVDV sequences included in GenBank, by comparing the differences between PDCoV and BVDV, design specific primers L1, L2, L3 and L4, as follows:
[0041] L1: 5'-ATCTCCCTAGCTTCGCTAGTTCTCTA-3' (SEQ ID NO.1)
[0042] L2: 5'-ATAGCGTCGCGCTTGGACTTGTTC-3' (SEQ ID NO.2)
[0043] L3: 5'-GGTGTGGAGGAACCTGTTTATGATCAG-3' (SEQ ID NO. 3)
[0044] L4: 5'-CCTTTACTATTACCCGACCTGCAGTCACCTC-3' (SEQ ID NO.4)
[0045] Using primers L1, L2, L3 and L4 for PCR amplification, the expected amplified fragment size of PDCoV is 525bp, and the expected amplified fragment size of BVDV is 185bp.
[0047] (1) Take PDCoV and BVDV porcine small intestine tissue of about 3 cm, after repeated freezing and thawing 3 times, cut it into pieces with high-pressure scissors, add 3 mL of high-pressure-treated PBS, grind it fully in a mortar, centrifuge the mixture at 13,000 g for 2 min, and take supernatant for later use; ...
Embodiment 2
[0075] The optimization of embodiment 2 primers
[0076] PDCoV primer optimization:
[0077] According to the PDCoV and BVDV sequences included in GenBank, by comparing the differences between PDCoV and BVDV, we designed PDCoV-specific primers L5 and L6, as follows:
[0078] L5: 5'-ATCAGCTGCTACCTCTC-3' (SEQ ID NO.5)
[0079] L6: 5'-TGGCTCATAGGTCTGGTTA-3' (SEQ ID NO.6)
[0080] Use primers L5 and L6 to perform PCR amplification at an annealing temperature of 51-60°C. The expected amplified fragment size of PDCoV is 516bp. See the results Figure 7 . Figure 7 It can be seen that the target band appears when the annealing temperature is 52°C, which is inconsistent with the annealing temperature of BVDV, and cannot be amplified in the same PCR reaction program, so the simultaneous detection of the two viruses cannot be completed.
[0081] In addition, the present invention also designs specific primers L7 and L8 to perform PCR amplification on PDCoV, and the specific sequence...
Embodiment 4
[0096] Example 4 specificity
[0097] Porcine circovirus, porcine blue ear disease virus, porcine pseudorabies virus, swine fever virus, porcine parainfluenza virus, porcine parvovirus, and porcine rotavirus were used as control strains, and PDCoV and BVDV were used as experimental strains. The method in example 1 detects ( Figure 12 ), the results showed that only PDCoV and BVDV could obtain 525bp and 185bp fragments respectively, and no amplification bands were produced by other viruses. Experimental results prove that the primer and detection method of the present invention have high specificity.
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Abstract
The invention discloses a multiple PCR (PolymeraseChain Reaction) detection primer set and kit for rapidly distinguishing a PDCoV (Porcine deltacoronavirus) and BVDV (Bovine Viral DiarrheaVirus). The primer set is a specific primer self-designed by an inventor according to PDCoV and BVDV sequences recorded in a GenBank through comparing differences between the PDCoV and the BVDV; and the primerset can carry out specific amplification on the PDCoV and the BVDV. On this basis, the inventor successfully develops the specific kit; firstly, virusRNA is extracted, and by adopting a RT-PCR (Reverse Transcription-PolymeraseChain Reaction) method, the PDCoV and the BVDV are distinguished and detected; the size of a PDCoV expected amplified fragment is 525bp; and the size of a BVDV expected amplified fragment is 185bp. Compared with the prior art, the specific kit disclosed by the invention has very high sensitivity, has the lowest detection capacity of 1.0*10<3> copies per muL and 1.2*10<4> copies per muL respectively for the PDCoV and the BVDV, has high specificity and good repeatability and can be widely used for clinical PDCoV and BVDV detection.
Description
technical field [0001] The invention relates to a diagnostic technique in the field of veterinary biotechnology, in particular to a multiplex PCR detection primer set and a kit for rapidly distinguishing porcine deltacoronavirus and porcine bovine viral diarrheavirus. Background technique [0002] Porcine deltacoronavirus (Porcine deltacoronavirus, PDCoV) is a newly emerged porcine coronavirus, belonging to Nidovirales (Nidovirales), Coronaviridae (Coronaviridae), and Coronavirus (Coronavirus), as a single-stranded positive chain Enveloped RNAvirus that was first detected in pigs in Hong Kong in 2012. Subsequently, the virus was also reported to be detected in the United States, Canada, and South Korea. The virus was first reported and detected in mainland China in 2014. Bovine viral diarrhea virus (BVDV) was first reported in the United States in 1946. my country first reported and isolated the virus in 1980. In addition to infecting cattle, BVDV can also infect wild...
Claims
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