Two-color fluorescence PCR primer, probe and method for quickly distinguishing canine parvovirus vaccine strain from wild strain

A canine parvovirus, two-color fluorescence technology, applied in the field of identification of virus vaccine virus and wild virus strain, can solve the problems of limited application, time-consuming, complicated operation, etc., and achieves the effects of good repeatability, reduced time, and simple operation.

Active Publication Date: 2016-08-10
INST OF ANIMAL HEALTH GUANGDONG ACADEMY OF AGRI SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The shortcomings of these methods have been described in detail above, and they all have shortcomings such as ...

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  • Two-color fluorescence PCR primer, probe and method for quickly distinguishing canine parvovirus vaccine strain from wild strain
  • Two-color fluorescence PCR primer, probe and method for quickly distinguishing canine parvovirus vaccine strain from wild strain
  • Two-color fluorescence PCR primer, probe and method for quickly distinguishing canine parvovirus vaccine strain from wild strain

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 primer pairs F1 / R1, F2 / R2, and probes P1 and P2 had the best effect on distinguishing canine parvovirus vaccine strains from wild strains by two-color fluorescence method. 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, two-color fluorescent PCR amplification and melting curve analysis

[0097] 1) Extraction of canine parvovirus DNA

[0098] Take samples from diseased materials infected with CPV. The samples of diseased materials can be whole blood, feces, rectal swabs and other samples that are easy to obtain and have no serious harm to the animal body. Take a certain amount and dissolve it in 1mL PBS hydrochloric acid buffer solution, let stand for 10-20min, take 200μl for later use; for CPV vaccine, add 3mL PBS hydrochloric acid buffer solution for dissolution, and take 200ul for later use. Nucleic acid extraction was carried out according to the instructions of TAKARA's MiniBEST Viral RNA / DNA Extraction KitVer.4.0.

[0099] 2) Preparation of standard samples

[0100] In order to verify the feasibility and reliability of the method of the present invention, construct a standard positive sample (correctly sequenced), and provide a positive...

Embodiment 3

[0132] Embodiment 3 specificity experiment

[0133] Next, the detection method established by the present invention is used for specific detection.

[0134] Extract other common canine diarrhea virus nucleic acids, such as canine distemper virμs (CDV), canine adenovirus-2 (Canine adenovirμs type 2, CAV-2), canine coronavirus (Canine coronavirus, CCV), canine rotavirus Viruses (Canine rotavirus, CRV) and canine parainfluenza virus (Canine parainfluenza virus (Canine parainfluenza virμs, CPIV), the RNA of CDV, CCV, CRV and CPIV were reverse-transcribed into cDNA, and the cDNA of the above virus, the nucleic acid of CAV-2 and water were used as The PCR template is analyzed with the PCR amplification reaction and melting curve analysis method in the above-mentioned embodiment 2, and canine parvovirus wild strain CPV-2a, CPV-2b, CPV-2c, vaccine strain CPV-int, CPV- The standard sample of pfu was compared and analyzed, and the peak shape of the melting curve was as follows figure...

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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 present invention relates to a method for distinguishing virus vaccine virus from wild strains, in particular to a two-color fluorescence detection method and kit for rapidly distinguishing canine parvovirus vaccine strains from wild strains. The method is a self-quenching detection method based on two double-labeled 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 single-stranded small DNA virus, which is the main cause of acute hemorrhagic gastroenteritis in dogs and acute myocarditis in puppies. CPV-2 emerged in the late 1970s but was replaced by new antigenic variants within a few years. At present, the main domestic CPV strains are 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 in a certain place in Jiangsu by PCR-...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
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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