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Porcine epidemic diarrhea virus TaqMan fluorescent quantitative RT-PCR kit and detection method thereof

A porcine epidemic diarrhea and fluorescence quantitative technology, which is applied in the field of animal virology and molecular biology, can solve the problems of poor repeatability, low positive detection rate, and poor sensitivity, and achieve good repeatability, strong specificity, and repeatability. The effect of a small coefficient of variation

Inactive Publication Date: 2018-02-13
LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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Problems solved by technology

[0004] The object of the present invention is to provide a kind of porcine epidemic diarrhea virus TaqMan fluorescence quantitative RT-PCR kit and detection method thereof, aim to solve its sensitivity difference of the method for existing detection porcine epidemic diarrhea virus, positive detection rate is low, repetitive Poor performance, can not be well used in the diagnosis of porcine epidemic diarrhea virus clinical samples and the problem of epidemiological investigation

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  • Porcine epidemic diarrhea virus TaqMan fluorescent quantitative RT-PCR kit and detection method thereof
  • Porcine epidemic diarrhea virus TaqMan fluorescent quantitative RT-PCR kit and detection method thereof
  • Porcine epidemic diarrhea virus TaqMan fluorescent quantitative RT-PCR kit and detection method thereof

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[0036] Example: a TaqMan fluorescent quantitative RT-PCR kit for porcine epidemic diarrhea virus and its detection method.

[0037] 1. Primer and probe design

[0038] According to the sequence alignment results of porcine epidemic diarrhea virus (PEDV) N gene DNA Star software, two pairs of primers and two probes were designed using the probe primer design software Primer Express5.0, see Table 1 for details.

[0039] Table 1 PEDV primers and TaqMan probe sequences Tab.1 The sequence of PEDV primers and probes

[0040]

[0041] Note: The N gene sequence of PEDV AH2012 strain is used as a template

[0042] 2. Extraction of PEDV RNA

[0043]The extraction of PEDV RNA was carried out according to the instructions of the RNeasy Mini kit (50) QIAGen operation manual. Specific operation: Take 350 μL of virus liquid and add it to a 1.5ml centrifuge tube, add an equal volume of RLT and 3.5 μL of β-mercaptoethanol, let it stand on ice for 5-10 minutes, then quickly add 700 μL of ...

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Abstract

The invention discloses a porcine epidemic diarrhea virus TaqMan fluorescent quantitative RT-PCR kit and a detection method thereof. According to an N gene sequence of a porcine epidemic diarrhea virus, a specific primer and a TanMan probe are designed and synthesized; and by optimizing a fluorescent quantitative reaction condition, a TaqMan real-time fluorescent quantitative RT-PCR method for detecting PEDV (Porcine Epidemic Diarrhea Virus) is established, has good linear relationship in a range from 10<8> copies / microlitres to 10<2> copies / microlitres, has the sensitivity capable of detecting 10<2> copies / microlitres to the minimum, is high in sensibility, can specifically and rapidly detect the porcine epidemic diarrhea virus and is good in repeatability with variation coefficient of intra-batch or inter-batch smaller than 1 percent. By applying the method to detect 185 pig manure samples collected in 2015-2017, and the positive detection rate is 38.4 percent; and when a conventional RT-PCR method for detection and a controlled trial is realized, the positive detection rate is 26.3 percent.

Description

technical field [0001] The invention belongs to the technical field of animal virology and molecular biology, and in particular relates to a TaqMan fluorescent quantitative RT-PCR kit and a detection method for porcine epidemic diarrhea virus. Background technique [0002] Porcine epidemic diarrhea virus (PEDV) is a common virus that causes acute intestinal infectious diseases in pigs. It is easy to infect, has a high incidence, and has a certain seasonality. ), porcine delta coronavirus (PDCoV), porcine crest virus (PKV), porcine reproductive and respiratory syndrome virus (PRRSV) and foot-and-mouth disease virus (FMDV). [0003] There are many methods for porcine epidemic diarrhea virus detection at home and abroad, including ELISA, RT-PCR and fluorescent quantitative PCR, etc., but the sensitivity of different methods is different. Among the above methods, the TaqMan probe real-time fluorescent quantitative RT-PCR method has higher sensitivity and specificity than other ...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
CPCC12Q1/6851C12Q1/701C12Q2531/113C12Q2561/101C12Q2545/113
Inventor 刘新生张永光王永录方玉珍周鹏张巧玲
Owner LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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