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RT-PCR (Reverse Transcription-Polymerase Chain Reaction) method for detecting porcine reproductive and respiratory syndrome virus (PRRSV) classical strains, high-pathogenicity variant strains and NADC-30 strains

A porcine blue ear virus, RT-PCR technology, applied in biochemical equipment and methods, DNA / RNA fragments, recombinant DNA technology, etc., can solve the problems of long detection time and complicated operation, and achieve high sensitivity and few operation steps , the effect of saving time

Inactive Publication Date: 2017-06-20
SOUTHWEST UNIVERSITY FOR NATIONALITIES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] In view of this, the present invention aims at the problems of complex operation and long detection time when using a single pair of primers to amplify three subtypes by PCR respectively, and provides a method for detecting classic strains of porcine PRRS virus and highly pathogenic variant strains. , The RT-PCR method of NADC-30 strain can detect clinical samples suspected of having porcine blue ear virus, so as to be used for the diagnosis and epidemiological monitoring of porcine blue ear virus

Method used

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  • RT-PCR (Reverse Transcription-Polymerase Chain Reaction) method for detecting porcine reproductive and respiratory syndrome virus (PRRSV) classical strains, high-pathogenicity variant strains and NADC-30 strains
  • RT-PCR (Reverse Transcription-Polymerase Chain Reaction) method for detecting porcine reproductive and respiratory syndrome virus (PRRSV) classical strains, high-pathogenicity variant strains and NADC-30 strains
  • RT-PCR (Reverse Transcription-Polymerase Chain Reaction) method for detecting porcine reproductive and respiratory syndrome virus (PRRSV) classical strains, high-pathogenicity variant strains and NADC-30 strains

Examples

Experimental program
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Effect test

Embodiment 1

[0023] Example 1 Primer Design

[0024] According to the three subtype sequences of the PRRSV gene registered in NCBI (classic strain of porcine PRRS virus (EF536003), highly pathogenic variant strain (EF112445) and NADC-30 strain (JN654459)), the software design was applied Primer5.0 For the Nsp2 gene of PRRSV, specific primers that can distinguish classic strains of porcine PRRSV, highly pathogenic variant strains, and NADC-30 strains were used. All primers were synthesized by Shanghai Sangon Bioengineering Co., Ltd.

[0025] Upstream primer (Nsp2F primer): GACACCTCCTTTGATTGG, SEQ ID NO: 1;

[0026] Downstream primer (Nsp2R primer): GAGAGGAIGCAGACAAATC (where I represents T or C or A or G), SEQ ID NO: 2;

[0027] Using above-mentioned primer RT-PCR amplification, the three subtypes of PRRSV can be quickly identified and distinguished by the size of the agarose gel electrophoresis band, the specificity and sensitivity are high, and the operation is simple.

Embodiment 2

[0028] Embodiment 2 detects the RT-PCR method of porcine PRRS classic strain, highly pathogenic variant strain and NADC-30 strain

[0029] The method includes the following steps:

[0030] (1) RNA extraction of samples to be tested:

[0031] The blood samples to be tested are centrifuged at 8000g for 5 minutes, and the upper serum is taken for each use; the tissue and organ samples need to be mixed with sterilized double distilled water at a mass volume ratio of 1:5, centrifuged at 8000g for 5 minutes after grinding, and the supernatant is taken for later use.

[0032] (2) The extracted RNA was added to a reverse transcription kit purchased from Bao Bio Engineering (Dalian) Co., Ltd. for reverse transcription to obtain a cDNA template:

[0033] The reverse transcription step is: mix 4 μL of RNA template with No. 1 5×PrimeScript Buffer 4 μL, No. 2 PrimeScriptRT Enzyme Mix 1 μL, No. 4 Random 6mers 2 μL and No. 5 RNase Free dH 2 O 9 μL was mixed and put into a PCR machine for r...

Embodiment 3

[0037] The optimization of embodiment 3 annealing temperature and primer concentration and the mensuration of sensitivity

[0038] (1) Cloning transformation and recombinant positive plasmid: RT-PCR amplifies the target gene, and conducts electrophoresis observation and analysis. The positive samples screened by electrophoresis were purified by the PCR purification kit purchased from OMGA Company, and the purified PCR products were connected to the TMD-19T carrier, put in the metal bath for 12 hours, and then taken out. The obtained ligation product was added to competent cells (Escherichia coli DH5α strain), and then the liquid was added to 1 mL of LB liquid, placed in a 37°C shaker box, and then centrifuged at 5000r / min for 4min, 800 μL of the supernatant was discarded, and the remaining 200 μL was blown and mixed. Take the bacterial liquid and drop it on the medium of LB+AMP, and spread the bacterial liquid evenly on the medium with a spreader stick, and put it in a 37°C ba...

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Abstract

The invention discloses an RT-PCR (Reverse Transcription-Polymerase Chain Reaction) method for detecting porcine reproductive and respiratory syndrome virus (PRRSV) classical strains, high-pathogenicity variant strains and NADC-30 strains. A pair of primers are designed by applying Primer5.0 software through referring to a PRRSV Nsp2 gene sequence published in GenBank. The pair of primers can be used for detecting three subtypes with different amino acid quantities of a PRRSV through an RT-PCR method. The RT-PCR method disclosed by the invention has high sensitivity and good specificity, and can be used for successfully detecting the three subtypes of the PRRSV, such as the classical strains, the variant strains ad the NADC-30 strains, and can be used for detecting clinical samples with the suspected PRRSV, so that the RT-PCR method is used for diagnosing the PRRSV and carrying out epidemiological monitoring.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to a RT-PCR method for detecting classic strains, highly pathogenic variant strains and NADC-30 strains of porcine PRRS virus. Background technique [0002] Porcine reproductive and respiratory syndrome virus (PRRSV), also known as porcine blue ear disease, is characterized by abortion, stillbirth, weak fetus, mummified fetus, dyspnea, sepsis and high mortality in piglets and fattening pigs. The disease is ubiquitous all over the world and has caused serious economic losses to the pig industry in my country. In recent years, the variation phenomenon of PRRSV has attracted people's attention. Foreign studies have shown that there are obvious sequence differences among the isolates of the same genotype, and the variation of PRRSV is one of the important reasons that cause the disease to be difficult to control. [0003] At present, according to the antigenic cha...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
CPCC12Q1/686C12Q1/701C12Q2600/16C12Q2521/107C12Q2565/125
Inventor 张斌岳华汤承王远微覃思楠
Owner SOUTHWEST UNIVERSITY FOR NATIONALITIES
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