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Triple fluorescent quantitative PCR primer, probe and kit for identifying pseudorabies virus strains

A pseudorabies virus, fluorescent quantitative technology, applied in the field of a-K61 vaccine, can solve the problems of inability to distinguish between classic and mutant strains of PRV, time-consuming and labor-intensive problems

Active Publication Date: 2016-07-20
HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these methods cannot discriminate between classic and variant strains of PRV
Although virus isolation combined with DNA sequencing can be used to identify and distinguish different strains, this method is time-consuming and laborious

Method used

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  • Triple fluorescent quantitative PCR primer, probe and kit for identifying pseudorabies virus strains
  • Triple fluorescent quantitative PCR primer, probe and kit for identifying pseudorabies virus strains
  • Triple fluorescent quantitative PCR primer, probe and kit for identifying pseudorabies virus strains

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 Establishment of triple fluorescent quantitative PCR method for identification and detection of PRV classic strain, variant strain and Bartha-K61 vaccine strain

[0031] 1. Materials and methods

[0032] 1.1 Virus

[0033] Swine fever virus (Classicalswinefevervirus, CSFV) Shimen strain, porcine parvovirus (Porcineparvovirus, PPV) Z strain, porcine circovirus type 2 (Porcinecircovirustype2, PCV2) JXL strain, porcine reproductive and respiratory syndrome virus (Porcinereproductiveandporcinerespiratorysyndromevirus, PRRSV) HuN4 strain , PRVSC strain, TJ strain, HLJMDJ2013 strain, HeBLP2014 strain, BJKJZ2015 strain (variant strain) and Bartha-K61 (vaccine strain) are preserved by the present inventor (among which the pseudorabies virus TJ strain, HLJMDJ2013 strain, HeBLP2014 strain and BJKJZ2015 strain, are by the present invention Humans were isolated, identified, and preserved, and their partial sequences were uploaded to GenBank at the same time, with sequenc...

Embodiment 2 3

[0049] Example 2 Optimization of the conditions of triple fluorescent quantitative PCR method

[0050] Based on the triple fluorescence quantitative PCR method established in Example 1, the present invention further optimizes the triple fluorescence quantitative PCR conditions.

[0051] 1. Experimental method

[0052]Triple fluorescent quantitative PCR experiments were performed using the Mx3005p instrument (Stratagene, USA), and the concentrations of primers and probes were optimized by the value of the fluorescent signal (ΔRn). Experimental conditions are as follows: PCR reaction total volume is 20 μ L, FAM-PRV-Cla (shown in SEQIDNo.1), HEX-PRV-Var (shown in SEQIDNo.4) and Cy5-PRV-Bar (shown in SEQIDNo.6) ( 10 μM) each 0.5 μ L; PRV-F1 (shown in SEQIDNo.8), PRV-R1 (shown in SEQIDNo.9), PRV-F2 (shown in SEQIDNo.14) and PRV-R2 (shown in SEQIDNo.15) ( Genomic DNA 2 μL; PremixExTaqProbeqPCR (TaKaRa, Japan) 10 μL; meanwhile, 0%, 2.5%, 5% and 10% (v / v) dimethyl sulfoxide (DMSO) w...

Embodiment 3 3

[0057] Specificity, sensitivity and repeatability test of embodiment 3 triple fluorescence quantitative PCR method

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Abstract

The invention discloses a triple fluorescent quantitative PCR primer, probe and kit for identifying pseudorabies virus classical strains, variant strains and Bartha-K61 vaccine strains. The sequences of the primer are represented by SEQ ID No. 8-9 and SEQ ID No. 14-15, and the sequences of the probe are represented by SEQ ID No. 1, 4 and 6. According to the kit, the pseudorabies virus classical strains, the variant strains and the Bartha-K61 vaccine strains can be accurately identified based on TaqMan probes marked with different fluorescence signals, and the kit has good sensitivity and specificity and can be applied to the discriminative detection of pseudorabies virus strains.

Description

technical field [0001] The invention relates to primers and probes for triple fluorescent quantitative PCR for identifying classic strains, mutant strains of pseudorabies virus and Bartha-K61 vaccine strains, and also relates to a method for identifying classic strains, mutant strains and Bartha-K61 vaccine strains of pseudorabies virus The triple fluorescence quantitative PCR detection kit belongs to the field of detection and identification of different strains of pseudorabies virus. Background technique [0002] Pseudorabies (PR), also known as Oyecki's disease, is an acute infectious disease of livestock and wild animals caused by Pseudorabies virus (PRV) infection, and is one of the pathogens that cause major economic losses in the world's pig industry. . Pigs are the reservoir host and main source of infection of the disease, and the clinical symptoms are mainly characterized by fever, nervous system and respiratory disturbances. Lactating piglets and young pigs are ...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
Inventor 仇华吉孟星宇罗玉子孙元
Owner HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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