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A two-color fluorescent detection method, primers and probes for rapidly distinguishing prv Chinese type, European and American type and vaccine strains

A two-color fluorescence and fluorescence detection technology, applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., can solve the problems of antibody detection lag, inability to identify strain types, unfavorable vaccine immunity, etc. Achieve high-throughput detection speed, fast detection speed, and good repeatability.

Active Publication Date: 2020-03-31
INST OF ANIMAL HEALTH GUANGDONG ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The gpI (gE) antibody ELISA test kit and the gB antibody ELISA test kit established based on the deletion of all or part of the gE gene in gene-deficient vaccines can achieve the purpose of identifying vaccine virus infection, but there is a serious lag in antibody detection. At the same time, it is impossible to identify the strain type, which is not conducive to further guiding vaccine immunization. Therefore, there is an urgent need for a method that is relatively simple to operate, reliable in detection results, and capable of high-throughput and rapid differentiation of different genotypes and vaccine strains of porcine pseudorabies virus.

Method used

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  • A two-color fluorescent detection method, primers and probes for rapidly distinguishing prv Chinese type, European and American type and vaccine strains
  • A two-color fluorescent detection method, primers and probes for rapidly distinguishing prv Chinese type, European and American type and vaccine strains
  • A two-color fluorescent detection method, primers and probes for rapidly distinguishing prv Chinese type, European and American type and vaccine strains

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1 Primers and Probes

[0048] After screening a large number of designed primers and probes, it was found that the primer pair F, R, and probes P1 and P2 had the best effect on distinguishing porcine pseudorabies virus Chinese type, European and American type and vaccine strain Bartha-K61 by two-color fluorescence method. Well, its base sequence is as follows.

[0049] Primer F: 5'-GGGCGTCTACACGTGGCGC-3' (SEQ ID NO: 1),

[0050] Primer R: 5'-GTTGGTCACGAAGGCGGCGT-3' (SEQ ID NO: 2),

[0051] Probe P1: 5'-FAM-CGCAGCGCCAACGTCTCGCTCGTCCTGTAC-BHQ1-3' (SEQ ID NO: 3),

[0052] Probe P2: 5'-HEX-CCGAGTTCGGCCTGAGCGCGCCGCC-BHQ1-3' (SEQ ID NO: 4).

Embodiment 2

[0053] Example 2 Preparation of standard samples, two-color fluorescent PCR amplification and melting curve analysis

[0054] 1) Extraction of porcine pseudorabies virus DNA:

[0055] Samples of diseased materials suspected of being infected with PRV were taken respectively. The samples of diseased materials could be lymph nodes, brain, heart, liver, spleen, lung, kidney, tonsil and other tissues of dead pigs, or collected pig serum or nose samples. Serum can be taken directly in 200 μl for later use; the nose formula needs to be dissolved in 1mL PBS hydrochloric acid buffer solution, and 200 μl of it should be taken after standing for 10-20 minutes; Add 3mL PBS hydrochloric acid buffer solution to dissolve, take 200μL for later use. Nucleic acid extraction was carried out according to the instructions of TAKARA's MiniBEST Viral RNA / DNA Extraction Kit Ver.4.0.

[0056] 2) Preparation of standard samples:

[0057] In order to verify the feasibility and reliability of the met...

Embodiment 3

[0081] Embodiment 3 specificity experiment

[0082] Next, the detection method established by the present invention is used for specific detection.

[0083]Extract other common porcine reproductive disorders related viral nucleic acids, such as extraction of classical swine fever virus (classical swine fever virus, CSFV), porcine encephalitis virus (Japanese encephalitis virus, JEV), porcine circovirus type 2 (Porcine circovirus type 2 , PCV 2), porcine reproductive and respiratory syndrome virus (Porcine Reproductive and Respiratory Syndrome Virus, PRRSV), porcine parvovirus (Porcine parvovirus, PPV), the RNA of CSFV, JEV and PRRSV was reverse transcribed into cDNA, and the cDNA of the above virus , the nucleic acid and water of PCV 2 and PPV were used as PCR templates respectively, analyzed by the PCR amplification reaction and melting curve analysis method in (2) above, and tested with the porcine pseudorabies virus Bartha-K61 vaccine strain (also European and American type...

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Abstract

A double-colour fluorescence detecting method, primers and probes for rapidly distinguishing a Genotype II porcine pseudorabies virus, a Genotype I porcine pseudorabies virus and a vaccine strain Bartha-K61 are disclosed. The method combines a real-time fluorescence PCR technique and a melting curve analysis technique. The Genotype II porcine pseudorabies virus, the Genotype I porcine pseudorabies virus and the vaccine strain Bartha-K61 are identified according to melting curve Tm value differences. Operation is simple, wherein identification detection of the two genotypes and the vaccine strain can be achieved by only one reaction. The detection speed is high in and the throughput is high. The total operation process can be finished in 3 hours. Virus cell culturing is not needed. Time for identification detection of the Genotype II porcine pseudorabies virus, the Genotype I porcine pseudorabies virus and the vaccine strain Bartha-K61 is greatly reduced. The method, the primers and the probes are high in accuracy, good in specificity, good in repeatability and capable of accurately and rapidly analyzing with a high throughput, and facilitate popularization and application in the clinical practice.

Description

technical field [0001] The present invention relates to different genotypes of viruses, and a method for identifying vaccine viruses and wild strains, in particular to a two-color fluorescence detection method for rapidly distinguishing Chinese type, European and American type of porcine pseudorabies virus (PRV), and vaccine strain Bartha-K61 And a kit, the method is a probe melting curve analysis technology based on two double-labeled self-quenching probes, which is used for the detection of porcine pseudorabies virus Chinese type, European and American type and vaccine strain Bartha-K61. Background technique [0002] Since 2011, large-scale pig farms in North China, Central China, and South China have successively experienced outbreaks of pseudorabies after being immunized with porcine pseudorabies vaccine. The positive rate of wild virus antibodies in some pig farms is as high as 100%, and 35% of pregnant sows have miscarriages. This is a major impact on my country's hog i...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/686C12N15/11
CPCC12Q1/686C12Q1/701C12Q2563/107C12Q2527/107
Inventor 张建峰刘志成孙俊颖张春红沈海燕
Owner INST OF ANIMAL HEALTH GUANGDONG ACADEMY OF AGRI SCI
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