Multiple fluorescent quantitative RT-PCR (reverse transcriptase-polymerase chain reaction) method for detecting PRRSV (porcine reproductive and respiratory syndrome virus) and application thereof

A TJM-PRRSV-P, multiple fluorescence quantitative technology, applied in the direction of fluorescence/phosphorescence, biochemical equipment and methods, microorganism determination/inspection, etc., can solve the problem of not being able to detect and distinguish at the same time

Inactive Publication Date: 2014-04-16
广西壮族自治区动物疫病预防控制中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, none of the established technologies can simultaneously detect and distinguish PRRSV American classic strains, variant strains and TJM-F92 vaccine strains

Method used

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  • Multiple fluorescent quantitative RT-PCR (reverse transcriptase-polymerase chain reaction) method for detecting PRRSV (porcine reproductive and respiratory syndrome virus) and application thereof
  • Multiple fluorescent quantitative RT-PCR (reverse transcriptase-polymerase chain reaction) method for detecting PRRSV (porcine reproductive and respiratory syndrome virus) and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] A multiplex fluorescent quantitative RT-PCR method for detecting PRRSV, the amplification reaction system in the RT-PCR method includes the following primers, probes and related reagents, see Table 3 for details. Primers and probes were designed by our laboratory, and other reagents can be purchased from the market. During the operation, positive control samples (standard products) with multiple concentration gradients are set up for RT-PCR amplification reaction to obtain a standard curve (control curve), and the RT-PCR amplification reaction of the detection sample is different from that of the positive control sample RT-PCR amplification reaction. Amplification reactions were performed simultaneously in different reaction wells by RT-PCR.

[0044] Table 3 Multiplex fluorescent quantitative RT-PCR reaction system for detecting PRRSV

[0045] Reaction system components Dosage (μL) Premix Ex Taq TM (Perfect Real Time) 12.5 AM-PRRSV upstream prime...

Embodiment 2

[0049] A multiplex fluorescent quantitative RT-PCR method for detecting PRRSV, the amplification reaction system in the RT-PCR method includes the following primers, probes and related reagents as in Table 4 in the examples. During the operation, positive control samples (standard products) with multiple concentration gradients are set up for RT-PCR amplification reaction to obtain a standard curve (control curve), and the RT-PCR amplification reaction of the detection sample is different from that of the positive control sample RT-PCR amplification reaction. Amplification reactions were performed simultaneously in different reaction wells by RT-PCR.

[0050] Table 4 Multiplex fluorescent quantitative RT-PCR reaction system for detecting PRRSV in this example

[0051] Reaction system components Dosage (μL) Premix Ex Taq TM (Perfect Real Time) 12.5 AM-PRRSV upstream primer (25pmol / μL) 0.2 AM-PRRSV downstream primer (25pmol / μL) 0.2 AM-V-PRRSV-P...

Embodiment 3

[0054] A multiplex fluorescent quantitative RT-PCR method for detecting PRRSV, the amplification reaction system in the RT-PCR method includes the following primers, probes and related reagents as in Table 5 in the examples. During the operation, positive control samples (standard products) with multiple concentration gradients are set up for RT-PCR amplification reaction to obtain a standard curve (control curve), and the RT-PCR amplification reaction of the detection sample is different from that of the positive control sample RT-PCR amplification reaction. Amplification reactions were performed simultaneously in different reaction wells by RT-PCR.

[0055] Table 5 Multiplex fluorescent quantitative RT-PCR reaction system for detecting PRRSV in this example

[0056] Reaction system components Dosage (μL) Premix Ex Taq TM (Perfect Real Time) 12 AM-PRRSV upstream primer (25pmol / μL) 0.3 AM-PRRSV downstream primer (25pmol / μL) 0.3 AM-V-PRRSV-P p...

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Abstract

The invention discloses a multiple fluorescent quantitative RT-PCR (reverse transcriptase-polymerase chain reaction) method for detecting PRRSV (porcine reproductive and respiratory syndrome virus) and an application thereof. The method is implemented by fluorescent quantitative RT-PCR primers and probes for detecting the PRRSV, wherein the primers comprise an AM-PRRSV primer pair and a TJM-PRRSV primer pair, and the probes comprise an AM-V-PRRSV-P probe, an AM-C-PRRSV-P probe and a TJM-PRRSV-P probe; and the multiple fluorescent quantitative RT-PCR method for detecting the PRRSV comprises the steps as follows: the primers and the probes are utilized to perform fluorescent quantitative RT-PCR amplification, fluorescence signals are collected after the amplification, the fluorescence signals which have amplification curves are positive, and the method is characterized in that an RT-PCR amplification system comprises the primers and the probes. The multiple fluorescent quantitative RT-PCR method can detect and distinguish PRRSV American type classical strains, HP-PRRSV and highly-pathogenic PRRS living-vaccine TJM-F92 strains simultaneously, and has the advantages of high specificity, high sensibility and high degree of automation.

Description

technical field [0001] The invention belongs to the technical field of molecular biological detection of animal viruses, and in particular relates to a multiplex fluorescent quantitative RT-PCR method for detecting porcine reproductive and respiratory syndrome virus (PRRSV), and its application to simultaneous detection of PRRSV classic strains of the American type and the American type. Rapid detection of mutant strains and highly pathogenic PRRS live vaccine TJM-F92 strain. Background technique [0002] Porcine reproductive and respiratory syndrome (PRRS), also known as "pig blue ear disease", is a highly Contact infectious diseases, clinically mainly manifested as reproductive disorders such as abortion, premature birth, stillbirth, mummified fetuses in pregnant sows, and severe respiratory diseases in pigs of various ages, especially piglets. PRRS first occurred in the United States in 1987, PRRSV was first isolated in the Netherlands in 1991, and the virus was isolated...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68G01N21/64
CPCC12Q1/686C12Q1/70C12Q2537/143C12Q2545/114C12Q2561/101
Inventor 施开创莫胜兰胡杰邹联斌张步娴屈素洁陆文俊粟艳琼苏凯李军
Owner 广西壮族自治区动物疫病预防控制中心
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