Dual real-time fluorescent RT-PCR identification and detection method of PRRSV classic strain and highly pathogenic mutant strain

A highly pathogenic, classic strain technology, applied in the biological field, can solve the problems of co-infection, time-consuming and labor-intensive, low sensitivity, etc., and achieve the effect of reducing detection cost, improving detection efficiency, and good reliability

Active Publication Date: 2010-02-10
CHINA ANIMAL DISEASE CONTROL CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the commonly used methods for detecting PRRSV mainly include virus isolation and identification, serological detection and common RT-PCR, but these conventional methods are time-consuming, laborious, low-sensitivity and prone to false positives
Real-time fluorescent RT-PCR detection methods for all PRRSV have been established at home and abroad, but this method can only detect whether there is PRRSV infection, and cannot determine whether it is a classic PRRSV infection, or a highly pathogenic variant of PRRSV infection, or both. co-infection
In addition, the existing real-time fluorescent RT-PCR detection method for highly pathogenic variants of PRRSV can only detect highly pathogenic variants of PRRSV, and cannot determine whether it is a single highly pathogenic variant of PRRSV or a classic strain of PRRSV. Co-infection with highly pathogenic variants
Even if the two existing PRRSV real-time fluorescent RT-PCR methods are used in combination, it is still impossible to determine whether there is a co-infection problem, and cannot provide strong technical support for the rapid and effective monitoring of the PRRSV epidemic in my country

Method used

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  • Dual real-time fluorescent RT-PCR identification and detection method of PRRSV classic strain and highly pathogenic mutant strain
  • Dual real-time fluorescent RT-PCR identification and detection method of PRRSV classic strain and highly pathogenic mutant strain
  • Dual real-time fluorescent RT-PCR identification and detection method of PRRSV classic strain and highly pathogenic mutant strain

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Experimental program
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Effect test

Embodiment 1

[0037] Construction of standard RNA

[0038] (1) Experimental reagents

[0039] The restriction enzyme Pst I, the corresponding buffer 10×H Buffer and DL2000Marker were purchased from TaKaRa Company;

[0040] -T Easy ligation kit, Taq DNA polymerase, AMV reverse transcriptase, RNase inhibitor, dNTPs were purchased from Promega;

[0041] DNA gel recovery kit, purchased from OMEGA company;

[0042] High-efficiency Escherichia coli competent cells DH5α were purchased from Beijing Quanshijin Biotechnology Co., Ltd.;

[0043] Primers were synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd.;

[0044] Other biochemical reagents are imported subpackages or domestic analytical pure.

[0045] (2) Experimental equipment

[0046] TGRANDIENT PCR instrument: purchased from HYBAID company;

[0047] DYFIII2 electrophoresis instrument: purchased from Bio-Rad;

[0048] UVP gel imaging analysis system: purchased from Gene Company;

[0049] Steril Biological saf...

Embodiment 2

[0064] Extraction of sample RNA

[0065] According to the manufacturer's recommendations, according to the routine method, using QIAGEN Mini Kit kit (purchased from QIAGEN), or according to the GTC method (guanidine isothiocyanate method) ( The Total RNA Isolation System kit was purchased from Promega) to extract RNA from samples to be tested from various sources.

Embodiment 3

[0067] Screening test of specific primers and probes for classic PRRSV strains and highly pathogenic variant strains by real-time fluorescent RT-PCR

[0068] (1) Experimental reagents

[0069] One Step PrimeScript TM RT-PCR Kit (Perfect Real Time) kit was purchased from TaKaRa Company;

[0070] The TaqMan MGB probes and primers for classic strains of PRRSV and highly pathogenic variants were synthesized by Shanghai Jikang Biotechnology Co., Ltd.

[0071] (2) Experimental equipment

[0072] Applied Biosystems 7500 Real-Time PCR System

[0073] (3) Experimental process

[0074] ① Design of primers and probes for classic PRRSV strains and highly pathogenic variant strains

[0075] The specific primers for classic PRRSV strains and highly pathogenic variant strains refer to: oligonucleotide chains with a length of about 20 bases, which are highly conserved with the ORF1a gene of classic PRRSV strains and highly pathogenic variant strains The specific nucleotide fragments ar...

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Abstract

The invention discloses a dual real-time fluorescent RT-PCR method for identifying and detecting a PRRSV classic strain and a highly pathogenic mutant strain, and an RT-PCR cation primer and a probe against the PRRSV classic strain and the highly pathogenic mutant strain for the method. Compared with the prior art, the method is characterized by rapidness, simplicity, strong specificity, high sensitivity and good reliability and can simultaneously carry out large batch sample analysis, and one-time reaction can identify and diagnose whether a sample is infected by the PRRSV classic strain or infected by the PRRSV highly pathogenic mutant strain or infected by the two strains, thereby providing a powerful technical support for monitoring and controlling a PRRS epidemic disease and having good application prospects.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for simultaneously detecting classical porcine reproductive and respiratory syndrome virus strains and highly pathogenic variant strains by using specific primers and probes. Background technique [0002] Porcine reproductive and respiratory syndrome (PRRS) is a contagious disease caused by porcine reproductive and respiratory syndrome virus (PRRSV). PRRSV was first isolated in the Netherlands and the United States, and served as the prototype strains of the European type (LV) and the American type (VR-2332), respectively. At present, PRRS has spread all over the countries and regions with developed pig industry in the world, and has brought huge economic losses to the world's pig industry and related industries. In recent years, PRRSV CH-1a, S1, HB-1 and HB-2 strains have been isolated successively in my country, all of which were identified as American PRRSV. Since June ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12N15/11C12R1/93
Inventor 田克恭陈南华陈西钊遇秀玲王传彬曹振
Owner CHINA ANIMAL DISEASE CONTROL CENT
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