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Fluorescent quantitative RT-PCR primers, probes and methods for detecting prrsv

A EU-PRRSV-P, AM-V-PRRSV-P technology, applied in biochemical equipment and methods, fluorescence/phosphorescence, microbial determination/inspection, etc., can solve problems such as inability to detect and distinguish at the same time

Inactive Publication Date: 2017-03-29
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 these techniques can simultaneously detect and distinguish PRRSV American-type classic strains, variant strains, and European-type strains

Method used

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  • Fluorescent quantitative RT-PCR primers, probes and methods for detecting prrsv
  • Fluorescent quantitative RT-PCR primers, probes and methods for detecting prrsv

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] A 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 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 primer (25pmol / µL) 0....

Embodiment 2

[0049]A 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 Fluorescence quantitative RT-PCR reaction system for detecting PRRSV in this example

[0051] Reaction system components Dosage (μL) Premix Ex Taq TM (Perfect Real Time)

[0052] The AM-PRRSV upstream primer specifically used in the RT-PCR amplification reaction system of this example has a sequence of 5'-G...

Embodiment 3

[0054] A 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 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)

[0057] The AM-PRRSV upstream primer specifically used in the RT-PCR amplification reaction system of this example has a sequence of 5'-G...

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Abstract

The invention discloses fluorescent quantitative RT-PCR (reverse transcriptase-polymerase chain reaction) primers, probes and a method for detecting PRRSV (porcine reproductive and respiratory syndrome virus). According to the fluorescent quantitative RT-PCR primers and probes for detecting the PRRSV, the primers comprise an AM-PRRSV primer pair and an EU-ORF1a primer pair, and the probes comprise an AM-V-PRRSV-P probe, an AM-C-PRRSV-P probe and an EU-PRRSV-P probe; and the fluorescent quantitative RT-PCR method for detecting the PRRSV comprises the steps as follows: the primers and the probes are utilized to perform multiple 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 fluorescent quantitative RT-PCR primers, the probes and the method for detecting the PRRSV can detect and distinguish PRRSV American type classical strains, HP-PRRSV and European type strains simultaneously, and has the advantages of high specificity, high sensibility, high degree of automation, speediness, simplicity, convenience and the like.

Description

technical field [0001] The invention belongs to the technical field of molecular biological detection of animal viruses, in particular to the application of multiple TaqMan fluorescent quantitative RT-PCR method to simultaneously detect the American type classic strain, the American type variant strain and the European type strain of porcine reproductive and respiratory syndrome virus (PRRSV). Primers, probes and methods for rapid detection. Background technique [0002] Porcine reproductive and respiratory syndrome (PRRS), also known as "porcine blue ear disease", is a highly contacted pig caused by porcine reproductive and respiratory syndrome virus (porcine reproductive and respiratory syndrome virus, PRRSV). Sexually transmitted 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 ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11G01N21/64
CPCC12Q1/686C12Q1/70C12Q2537/143C12Q2561/101C12Q2531/113
Inventor 施开创张步娴官家明邹联斌胡杰陆文俊莫胜兰屈素洁粟艳琼李军
Owner 广西壮族自治区动物疫病预防控制中心