Reverse transcription-polymerase chain reaction (RT-PCR) detection for porcine reproductive and respiratory syndrome virus and classical swine fever virus and special primers for same

A technology for RT-PCR and respiratory syndrome, which is applied in the field of RT-PCR detection and its special primers, can solve the problems of high detection cost, time-consuming and laborious, and not fast, and achieve strong sensitivity, good application prospects, and high specificity Effect

Inactive Publication Date: 2012-07-18
山东省动物疫病预防与控制中心
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Problems solved by technology

The commercial PRRSV and CSFV detection RT-PCR kits are all single-item RT-PCR. One kit can only detect one virus in one test. For the same pig disease that may be mixed with PRRSV, HP-PRRSV, and CSFV It is expected that 3

Method used

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  • Reverse transcription-polymerase chain reaction (RT-PCR) detection for porcine reproductive and respiratory syndrome virus and classical swine fever virus and special primers for same
  • Reverse transcription-polymerase chain reaction (RT-PCR) detection for porcine reproductive and respiratory syndrome virus and classical swine fever virus and special primers for same
  • Reverse transcription-polymerase chain reaction (RT-PCR) detection for porcine reproductive and respiratory syndrome virus and classical swine fever virus and special primers for same

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Embodiment 1

[0061] Embodiment 1, multiple RT-PCR detection method construction of porcine reproductive and respiratory syndrome virus, highly pathogenic porcine reproductive and respiratory syndrome virus and classical swine fever virus and design of special primers

[0062] 1. Primer design

[0063] In GenBank, HP-PRRSV (highly pathogenic porcine reproductive and respiratory syndrome virus, Genbank number EF112445), PRRSV (porcine reproductive and respiratory syndrome virus classic strain, Genbank number AY032626), CSFV (swine fever virus, Partially known sequence of Genbank No. AF092448). Homology analysis was performed on each gene region of the virus, and a segment of the NSP2 gene of PRRSV and the E2 gene of CSFV were respectively selected as the target sequence for PCR amplification. Using DNAMAN to design primers for the target sequence, the designed two virus primers (primer pair A can simultaneously amplify the classical strain of porcine reproductive and respiratory syndrome vi...

Embodiment 2

[0101] Embodiment 2, the application of special primer

[0102] 315 clinically suspected disease materials (from the mixed samples of liver, kidney and lung tissues (mass ratio 1:1:1) of dead pigs sent for inspection at the Veterinary Hospital Attached to the Animal Disease Prevention and Control Center of Shandong Province) were tested. as follows:

[0103] Extract 315 RNAs (numbered 1-315) of clinically suspected disease materials, use primer pair A and B as primers, and perform RT-PCR amplification according to the system and procedures in step 2,

[0104] If the RT-PCR product (sequence 5 in the sequence listing) of 525bp is obtained, the sample is positive for PRRSV;

[0105] If the RT-PCR product (sequence 6 in the sequence listing) of 435bp is obtained, the sample is positive for HP-PRRSV;

[0106] If the RT-PCR product (sequence 7 in the sequence listing) of 707bp is obtained, the sample is positive for CSFV;

[0107] If RT-PCR products of 525bp and 435bp are obtain...

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Abstract

The invention discloses reverse transcription-polymerase chain reaction (RT-PCR) detection for porcine reproductive and respiratory syndrome virus and classical swine fever virus and special primers for same. The invention provides the special primers for detecting the viruses. The special primers comprise a primer pair A and a primer pair B, wherein the primer pair A comprises a primer 1 and a primer 2; the primer pair B comprises a primer 3 and a primer 4; and nucleotide sequences of the primers 1, 2, 3 and 4 are sequences 1, 2, 3 and 4 in a sequence table. Experiments prove that: by the multiplex RT-PCR detection method for the porcine reproductive and respiratory syndrome virus and the classical swine fever virus, a classical porcine reproductive and respiratory syndrome virus strain, a high pathetic porcine reproductive and respiratory syndrome virus strain and the classical swine fever virus can be simultaneously detected through once reaction, and the method has a good application prospect on clinical diagnosis.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to RT-PCR detection of porcine reproductive and respiratory syndrome virus and swine fever virus and special primers thereof. Background technique [0002] The RT-PCR detection method of porcine reproductive and respiratory syndrome virus (PRRSV) and classical swine fever virus (CSFV) is currently a common method for rapid detection of these two pathogens, and is widely used in laboratory pathogen diagnosis. Compared with the virus isolation method, the RT-PCR method is faster and easier to diagnose, and the virus isolation is relatively time-consuming, which is not conducive to rapid clinical diagnosis. At present, the laboratory diagnosis of viral diseases mainly uses the PCR / RT-PCR method. The RT-PCR kits of PRRSV and CSFV have been commercialized and put into clinical application. [0003] The current multi-virus mixed infection of swine disease is quite serious. Clinically, porci...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
Inventor 李云岗杜兰荣张栋孙圣福兰邹然
Owner 山东省动物疫病预防与控制中心
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