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A two-color fluorescent PCR primer, probe and method for rapidly distinguishing canine parvovirus vaccine strains from wild strains

A canine parvovirus, two-color fluorescence technology, applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., can solve the problems of time-consuming, complicated operation, limited promotion and application, etc., to reduce the time , Good repeatability, high-throughput detection speed

Active Publication Date: 2019-12-24
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

The shortcomings of these methods have been described in detail above, and they all have shortcomings such as complicated operation and time-consuming, and their popularization and application in clinical practice are limited.

Method used

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  • A two-color fluorescent PCR primer, probe and method for rapidly distinguishing canine parvovirus vaccine strains from wild strains
  • A two-color fluorescent PCR primer, probe and method for rapidly distinguishing canine parvovirus vaccine strains from wild strains
  • A two-color fluorescent PCR primer, probe and method for rapidly distinguishing canine parvovirus vaccine strains from wild strains

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0088] Example 1 Primers and probes

[0089] After screening a large number of designed primers and probes, it was found that the primer pairs F1 / R1, F2 / R2, and probes P1 and P2 have the best effect on the two-color fluorescence method to distinguish canine parvovirus vaccine strains from wild virus strains. The base sequence is shown below.

[0090] F1: 5'-TATAGCACATCAAGATACAGGAAGA-3' (SEQ ID NO: 1),

[0091] R1: 5'-TCCAATTGGATCTGTTGG-3' (SEQ ID NO: 2),

[0092] Probe P1: 5'-FAM- CCTATCATCTTCCTGTAACAAATGATAG-BHQ1-3' (SEQ ID NO: 3),

[0093] F2: 5'-TGGAAATCACAGCAAACTC-3' (SEQ ID NO: 4),

[0094] R2: 5'-CTAAAGCCATGTTTCCGT-3' (SEQ ID NO: 5),

[0095] Probe P2: 5'-HEX-CGACCGTAAATAATATGGATAAAACTGCGGTCG-BHQ1-3' (SEQ ID NO: 6).

Embodiment 2

[0096] Example 2 Preparation of standard samples, dual-color fluorescence PCR amplification and melting curve analysis

[0097] 1) Extraction of canine parvovirus DNA

[0098] Take samples of disease material infected with CPV. Samples of disease material can be whole blood, feces, rectal swabs and other easy-to-obtain samples that do not cause serious harm to the animal. Whole blood can be directly taken 200μl for use. Stool and rectal test samples are required. Take a certain amount and dissolve it in 1mL PBS hydrochloric acid buffer solution, let it stand for 10-20 min and take 200μl for use; for CPV vaccine, add 3mL PBS hydrochloric acid buffer solution to dissolve, take 200μL for use. Nucleic acid extraction was carried out according to the instructions of MiniBEST Viral RNA / DNA Extraction Kit Ver.4.0 of TAKARA.

[0099] 2) Preparation of standard samples

[0100] In order to verify the feasibility and reliability of the method of the present invention, while constructing a stan...

Embodiment 3

[0132] Example 3 Specific Experiment

[0133] The following is a specific test for the detection method established in the present invention.

[0134] Extract nucleic acid from other common canine diarrhea viruses, such as Canine distemper virμs (CDV), Canine adenovirμs type 2 (CAV-2), Canine coronavirus (CCV), and canine rotavirus Virus (Canine rotavirus, CRV) and Canine parainfluenza virus (Canineparainflμenza virμs, CPIV), reverse transcription of CDV, CCV, CRV and CPIV RNA into cDNA, and use the cDNA of the above virus, the nucleic acid of CAV-2 and the water respectively as The PCR template was analyzed by the PCR amplification reaction and melting curve analysis method in Example 2 above, and compared with the wild canine parvovirus strains CPV-2a, CPV-2b, CPV-2c, vaccine strains CPV-int, CPV- The standard samples of pfu are compared and analyzed, and the melting curve peak pattern is shown as figure 2 Shown.

[0135] From figure 2 It can be seen from A~C that the detection...

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Abstract

The invention discloses a two-color fluorescence PCR primer, probe and method for quickly distinguishing canine parvovirus vaccine strain from wild strain. The real-time fluorescence PCR technique and the melting curve analysis technique are combined, and canine parvovirus vaccine strain and wild strain are identified according to the peak pattern of a melting curve and Tm value difference; operation is easy, and identification and detection of three kinds of wild strain with different genotypes and two kinds of vaccine strain can be achieved through one reaction; detection speed is high, and flux is high; the whole operation process can be finished within 3 h, cell cultivation of viruses is not needed, and time for identification and detection of three kinds of wild strain with different genotypes and two kinds of vaccine strain is shortened greatly; accuracy, specificity and repeatability are high, accurate, quick and high-flux analysis can be achieved, and application and popularization in clinical practice can be achieved easily.

Description

Technical field [0001] The invention relates to a method for identifying a virus vaccine virus and a wild virus strain, in particular to a two-color fluorescence detection method and a kit for quickly distinguishing a canine parvovirus vaccine strain from a wild virus strain. The method is a self-quenching detection method based on two double labels The needle probe melting curve analysis technology is used in the detection method of canine parvovirus wild strain and vaccine strain. Background technique [0002] Canine parvovirus (CPV) is a small single-stranded DNA virus, which is the main pathogen causing acute hemorrhagic gastroenteritis in dogs and acute myocarditis in puppies. CPV-2 appeared in the late 1970s, but was replaced by new antigen variants within a few years. At present, the main domestic CPV strain is CPV-2a / 2b. In 2010, Xia Xianzhu et al. reported the CPV-2c variant for the first time. In 2014, Gali Bingga et al. identified CPV-2c somewhere in Jiangsu through P...

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 陈琴苓刘志成张建峰嘎利兵嘎张春红魏文康
Owner INST OF ANIMAL HEALTH GUANGDONG ACADEMY OF AGRI SCI
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