Primer and probe combination for simultaneously detecting three infectious bovine pathogens

A probe and primer sequence technology, which is applied in the application field of the primer and probe combination, can solve the problems of lengthy operation, cumbersome operation, and long time, so as to improve accuracy and sensitivity, broad application space, and improve detection efficiency Effect

Pending Publication Date: 2022-07-22
HUAZHONG AGRI UNIV
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  • Description
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AI Technical Summary

Problems solved by technology

The isolation and identification of viruses is the gold standard, but this method is cumbersome to operate and takes a long time to diagnose; serum neutralization test can be used for virus antigen detection, which has strong specificity, but the operation is complicated and takes a long time; ELISA technology can also be used for virus antigen detection. Antigen detection is simple, fast, and specific, but lacks commercial kits and has low diagnostic sensitivity; traditional PCR technology cannot quantify template DNA, and it needs to be interpreted by gel electrophoresis in the end, and the operation is relatively lengthy , and the risk of contamination is high; real-time fluorescence quantitative PCR technology combines fluorescent substances with traditional PCR technology, not only can accurately identify and absolutely quantify pathogens, but also has simple operation, good specificity, high sensitivity, and can also dynamically study Resurrection or persistent infection of potential pathogens throughout the course of the disease; multiplex real-time fluorescent quantitative PCR technology can accurately and quickly detect mixed infections caused by various pathogens

Method used

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  • Primer and probe combination for simultaneously detecting three infectious bovine pathogens
  • Primer and probe combination for simultaneously detecting three infectious bovine pathogens
  • Primer and probe combination for simultaneously detecting three infectious bovine pathogens

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] The design of the primer of embodiment 1

[0036] All nucleotide sequences of BVDV-5'UTR; BRV-VP6; BCV-N gene region were downloaded from GenBank, and the MEGA software was used for alignment analysis to select the most conserved gene sequence (BVDV GenBank accession number: LC099927.1 ; BRV GenBank accession number: AB374146.1; BCV GenBank accession number: LC494138.1) and its specific region, designed and screened specific primers and probes (Table 1).

[0037] Table 1 Primers and probes

[0038]

Embodiment 2

[0039] Example 2 Establishment and optimization of multiplex real-time fluorescent quantitative PCR method

[0040] 1. Establishment and optimization of single detection real-time quantitative PCR method for BVDV, BRV and BCV pathogens

[0041] 1.1 Standard positive plasmid preparation

[0042] Using the primers in Table 1, BVDV-5'UTR; BRV-VP6; BCV-N gene region (BVDV GenBank Accession No.: LC099927.1; BRV GenBank Accession No.: AB374146.1; BCV GenBank Accession No.: LC494138.1) A segment of the sequence including the primer sequence was inserted into the pUC57 vector to form a recombinant plasmid, named pUC57-5'UTR, pUC57-VP6, pUC57-N as the standard positive plasmid template and positive control in the system construction.

[0043] 1.2 Establishment of single-detection real-time fluorescent quantitative PCR method

[0044] A single factor variable was set, and according to the primers and probes in Table 1, real-time quantitative PCR was used to optimize the primer concent...

Embodiment 3 3

[0094] Example 3 Evaluation of the triple real-time fluorescence quantitative PCR system for the detection of clinical samples

[0095] Using the triple real-time fluorescence quantitative PCR system established in this study and the common PCR detection method obtained from the literature review (Table 10), 143 cDNA samples stored in the laboratory were tested for clinical samples and the coincidence rate was analyzed. The results of each common PCR test were verified by sequencing of Wuhan Qingke Biotechnology Co., Ltd.; the criteria for the triple real-time PCR detection method were:

[0096] 1. The Ct value of the tested sample is less than 40 and it is judged to be positive when a typical amplification curve appears;

[0097] 2. When the Ct value of the tested sample is greater than 40 and there is a typical amplification curve, it needs to be re-tested. If the re-examination result is as above, it will be judged as positive, otherwise it will be judged as negative;

[...

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Abstract

The invention discloses a triple real-time fluorescent quantitative PCR (Polymerase Chain Reaction) primer and probe combination for simultaneously detecting BVDV (Bovine Viral Diarrhea Virus), BRV (Bovine Rotavirus) and BCV (Bovine Coronaviruses), the specificity of the primer and the specificity of the probe aim at the 5 'UTR gene segment of the BVDV, the VP6 gene segment of the BRV and the N gene segment of the BCV, and the nucleotide sequences of the primer and the probe are as shown in SEQ ID No.1-9. The invention further discloses a kit for detecting the BVDV, the BRV and the BCV. According to the invention, qualitative or quantitative detection of three viruses can be realized only by performing PCR amplification on a sample once, and the method has the advantages of simplicity and convenience in operation, strong specificity, high sensitivity, good repeatability and the like.

Description

technical field [0001] The invention belongs to the field of molecular detection, in particular to a set of primers and probes for simultaneous detection of bovine viral diarrhea virus, bovine rotavirus and bovine coronavirus, and also relates to the application of the primers and probes. [0002] technical background [0003] Bovine viral diarrhea virus (BVDV) is the causative agent of bovine viral diarrhea (BVD). BVDV virus infection shows a wide range of clinical manifestations, including acute hemorrhagic syndrome, long-term persistent virus Infection, weakened immunity, and severe respiratory and gastrointestinal lesions; the reproductive failure caused by BVDV infection is one of the most serious consequences. BVDV virus infection is mostly persistent infection. Some infected cattle are healthy in appearance and negative for antibodies, but the antigen of the virus is positive. Such persistently infected cattle carry the virus for life, which will cause great harm to th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/686C12N15/11
CPCC12Q1/701C12Q1/686C12Q2600/16C12Q2561/113C12Q2563/107C12Q2545/114C12Q2537/143
Inventor 郭爱珍马耀争胡长敏陈颖钰朱杰陈曦陈建国
Owner HUAZHONG AGRI UNIV
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