Porcine epidemic diarrhea virus (PEDV) fluorescent quantitative PCR primer and probe

A porcine epidemic diarrhea and fluorescence quantitative technology, applied in the field of molecular biology, can solve the problem of undetected mutant strains, and achieve the effects of fast detection speed, simple operation and high accuracy

Inactive Publication Date: 2017-07-14
HENAN ACAD OF AGRI SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The S gene is a characteristic gene for distinguishing different strains of PEDV. At present, a Taqman probe fluorescence quantitative PCR method based on the S gene has been reported to identify and detect different strains of PEDV (Chinese Patent No. CN103509882A). Due to the continuous variation of the gene, it is found that there is a base mutation in the probe sequence of the mutant strain described in this method. Due to the high specificity of the Taqman probe, the new mutant strain may not be detected

Method used

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  • Porcine epidemic diarrhea virus (PEDV) fluorescent quantitative PCR primer and probe
  • Porcine epidemic diarrhea virus (PEDV) fluorescent quantitative PCR primer and probe
  • Porcine epidemic diarrhea virus (PEDV) fluorescent quantitative PCR primer and probe

Examples

Experimental program
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Effect test

Embodiment 1

[0046] Example 1: Design and synthesis of probes and primers

[0047] The primers and probes are the classic strains of porcine epidemic diarrhea virus published in GenBank in recent years by the DNAMAN8 software (the accession numbers of the representative strains of the classic strains are: AF353511.1, JN547228.1, KT323979.1, EF185992. 1, JN601050.1, JX560761.1) and mutant strains (the accession numbers of the mutant strains are: KC210145.1, JX112709.1, KU133250.1, KJ646584.1, KP698764.2, KT428879.1, KX073609.1, KU133236.1, KU133259.1, KU237226.1, KU710232.1, KR233257.1, KJ646580.1, KU237230.1, KU237229.1, KU977512.1, KU710235.16U71.2K2, KU133 1, KU710234.1, KU710229.1, KU710242.1, KR296683.1, KT989356.1, KU237217.1) S1 gene sequence alignment, design two specific probes in the sequence difference region, and at the probe position Design common primers for the periphery, and then use MFEprimer-2.0 to evaluate the feasibility of probes and primers. All primers and probes we...

Embodiment 2

[0048] Example 2: RNA extraction and cDNA synthesis

[0049] Take 300 μl of the cell culture medium infected with the classic strain of porcine epidemic diarrhea virus CV777 (this strain has been identified as a classic strain of porcine epidemic diarrhea virus, and other classic strains can also be used instead), 300 μl of the cell culture medium infected with the variant virus of porcine epidemic diarrhea virus Strain CH / HNLY (this strain has been identified as a porcine epidemic diarrhea virus variant strain, and other variant strains can also be used instead) clinical sample processing supernatant (take the infected porcine epidemic diarrhea virus variant strain CH / HNLY Cut pig intestinal tissue into pieces with scissors, add appropriate amount of normal saline, freeze-thaw three times, centrifuge at 3000rpm / min for 5min to get the supernatant), add 800μl Trizol respectively, mix well, and let stand at room temperature for 10min; then add 200μl chloroform, fully Mix well, ...

Embodiment 3

[0051] Embodiment 3: gene cloning

[0052] Using the cDNA obtained by reverse transcription as a template, use the upstream and downstream primers (SEQ ID NO.1-SEQ ID NO.2) of the present invention to amplify the target fragment respectively, wherein the PCR amplification reaction system is: template 1 μl, 2× Ex Taq 10μl, upstream and downstream primers (10pmol) each 1μl, supplemented with deionized water to 20μl; PCR reaction program: 95°C pre-denaturation for 5min, and then the following 30 cycles: 95°C denaturation for 30s, 55°C for 30s, 72°C Extend for 30s, and then extend for 5min at 72°C after the cycle ends. The PCR amplified product was identified by 1% agarose electrophoresis, and the target fragment was recovered with a gel recovery kit (TaKaRa), and the target fragment was cloned into the Peasy-Blunt vector and transformed into DH5α competent cells. The positive clones were picked by blue-white screening and sequenced. Positive recombinant plasmids were identified....

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Abstract

The invention discloses a fluorescent quantitative PCR identification and detection method of a PEDV classical strain and a PEDV variant strain. According to the method, based on the S1 gene of a PEDV classical strain and a PEDV variant strain, two specific probes are designed, and a pair of specific primers is designed on the periphery of the probes. The cDNA of a representative strain CV777 of PEDV classical strain and a representative strain CH / HNLY of PEDV variant strain is taken as the target fragment of template amplification, recombinant plasmids are established, taken as the standard substance, and used to establish a standard curve, and according to different report genes of two probes, different strains are identified and detected. The provided method has good specificity, sensitivity, and repeatability, and can be used to identify and detect a PEDV sample in clinic.

Description

technical field [0001] The invention belongs to the field of molecular biology, and in particular relates to a fluorescent quantitative PCR primer and probe capable of identifying and detecting classic strains and variant strains of porcine epidemic diarrhea. Background technique [0002] Porcine epidemic diarrhea (PED) is an acute, highly contagious disease characterized by severe watery vomiting and diarrhea and dehydration caused by porcine epidemic diarrhea virus (PEDV) Enteroviral diseases mainly harm newborn piglets with a mortality rate as high as 100%. PEDV belongs to the Coronaviridae family, and the coronavirus belongs to group 1. It is a single-stranded positive-sense non-segmented RNA virus. The full-length 28-kb genome contains 7 open reading frames, and the four structural protein genes encoded are: S gene , E gene, M gene and N gene. PED was first discovered in the United Kingdom, and subsequently reported in many countries, such as Belgium, Japan, South Kor...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
CPCC12Q1/6851C12Q1/701C12Q2563/107C12Q2561/101C12Q2545/114
Inventor 张改平王爱萍苏运芳刘运超邓瑞广邢广旭陈玉梅郝慧芳王娟杨聪聪
Owner HENAN ACAD OF AGRI SCI
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