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PCR-HRM primer and detection method for rapid identification of PRRSV gene subtype and application of PCR-HRM primer

A technology of PCR-HRM and PRRSV-F, which is applied in the field of PCR-HRM primers for rapid identification of PRRSV genotypes, can solve the problems of inability to quickly and accurately identify PRRSV genotypes, inability to distinguish vaccine strains from wild strains, and high cost. Reliable identification, shortened time required and high repeatability are achieved

Pending Publication Date: 2019-11-12
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

Studies have found that the recombination of PRRSV vaccine strains and wild strains is very common. There are multiple PRRSV strains in a single pig herd. It is now impossible to identify vaccine strains and wild strains through single nucleotide differences, let alone rapid and accurate identification Genotypes of PRRSV in pig farms
At present, PRRSV genotyping needs to be completed by PCR amplification, sequencing and sequence analysis software, which takes 2-3 days, which is long and expensive

Method used

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  • PCR-HRM primer and detection method for rapid identification of PRRSV gene subtype and application of PCR-HRM primer
  • PCR-HRM primer and detection method for rapid identification of PRRSV gene subtype and application of PCR-HRM primer
  • PCR-HRM primer and detection method for rapid identification of PRRSV gene subtype and application of PCR-HRM primer

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Effect test

Embodiment 1

[0038] Embodiment 1 PCR-HRM primer design

[0039] The inventors of the present invention used PCR amplification and sequencing of the PRRSV GP5 gene in clinical samples of more than 100 large-scale pig farms in Guangdong Province, and used the sequence analysis software MEGA7 to draw the genetic evolution tree of PRRSV. The results are shown in figure 1. The characteristic sequences of the four genotype subtypes of PRRSV were found through the genotyping software, and multiple pairs of primers were designed and screened repeatedly to obtain primers that could efficiently amplify all strains of PRRSV. The length of the fragment amplified by the primers is 117 bp, which contains the differential gene sites of the four genotype subtypes of PRRSV, and this region is also the main antigenic site of neutralizing antibodies produced after PRRSV vaccine immunization. The nucleotide sequences of the primers are as follows:

[0040] PRRSV-F: 5'-TTGTGGTGTATCGTGCCRT-3' (SEQ ID NO: 1); ...

Embodiment 2

[0043] Preparation and PCR-HRM analysis of embodiment 2 standard samples

[0044] (1) Preparation of positive standard samples:

[0045] In order to verify the feasibility and reliability of the method of the present invention, the standard strains of 4 gene subtypes PCR-HRM analysis of PRRSV are established simultaneously, and subtype I and subtype II select the widely used vaccine strain JXA1-R strain ( (purchased from Guangdong Animal Vaccine Supply Station) and RespPRRS MLV strain (purchased from Guangdong Animal Vaccine Supply Station) were used as the standard strains of these two genotype subtypes. Two strains, GD-GM strain (published on GenBank, gene bank accession number: KX429681.1) and GD-HH strain (already published on GenBank) of the same genotype as NADC30 strain and GM2 strain, were isolated from The gene bank accession number is: KX429682.1) as the standard strain-positive samples of these two genotypes, providing a typing reference for PCR-HRM analysis for su...

Embodiment 3

[0056] The PCR-HRM detection of embodiment 3 clinical sample

[0057] (1) Extracting viral RNA from samples suspected of being infected with PRRSV, the method is the same as the RNA extraction method in Example 2.

[0058] (2) Using the extracted RNA as a template, perform reverse transcription and PCR-HRM amplification (one-step amplification). The amplification reaction system is: 2 μL of extracted RNA, 2 times the concentration of one-step buffer (2 × Step Buffer) 10 μL, primer PRRSV-F (10 μM) 0.5 μL, primer PRRSV-R (10 μM) 0.5 μL, PrimeScript 1 Step Enzyme Mix 1 μL, saturated fluorescent dye LC Green solution (diluted 20 times according to the instructions) 1 μL, ddH 2 O to make up to 20 μL.

[0059] The amplification reaction program was: reverse transcription at 50°C for 5min; pre-denaturation at 95°C for 2min; denaturation at 95°C for 10s, extension at 72°C for 35s, 45 cycles. The heating steps of the HRM program were 92°C for 1 min, 40°C for 2 min; from 60°C to 90°C,...

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Abstract

The invention discloses a PCR-HRM primer and detection method for rapid identification of PRRSV gene subtype and application of the PCR-HRM primer. The sequence of the primer is as shown in SEQ ID NO.1-2, the PCR-HRM primer has good amplification for PRRSV vaccine strains widely used in the market and epidemic strains in pig farms, the PCR amplification efficiency is high, the detection sensitivity is high, the specificity is good, and the repeatability is high. According to the provided PCR-HRM detection method, operation foranalyzing and identifying four PRRSV gene subtypes is easy, the whole process only needs 2 hours, and the time required by genotyping is greatly shortened; the cost is low, sequencing is not needed, a synthetic probe is also not needed, and cheap and easily availablefluorescence saturated dye only needs to be added. The PCR-HRM primer can be used for preparing a PRRSV subtype detection kit, PRRSV subtype identification in farms, molecular epidemiological investigation and PRRSV subtype risk assessment in aquaculture, and the application prospect is broad.

Description

technical field [0001] The invention belongs to the field of virus detection, and relates to a PCR-HRM primer for rapidly identifying PRRSV gene subtypes, a detection method and an application. Background technique [0002] Porcine Reproductive and Respiratory Syndrome (PRRS) was first discovered in the United States in 1987, and PRRSV was first isolated and identified in my country in 1996, confirming the existence of the disease in my country. Its symptoms are mainly manifested as abortion, premature birth, stillbirth, mummified fetus and decreased survival rate of infected piglets in pregnant sows, and respiratory symptoms in adult pigs. [0003] PRRSV is divided into two genotypes based on serological tests and gene sequences, namely the American type (the representative strain is VR-2332 and its vaccine strain RespPRRS MLV) and the European type (the representative strain is LV). type-based. The pathogen of the high fever outbreak in 2006 was the highly pathogenic PRR...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6858C12N15/11C12R1/93
CPCC12Q1/701C12Q1/6858C12Q2531/113C12Q2527/107C12Q2521/107Y02A50/30
Inventor 蒋智勇楚品品蔡汝健李春玲李艳宋帅勾红潮杨冬霞
Owner INST OF ANIMAL HEALTH GUANGDONG ACADEMY OF AGRI SCI
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