PDCoV detection method based on real-time fluorescence RT (reverse transcription) RAA (recombinase-aid amplification) technology

A real-time fluorescence, coronavirus technology, applied in the field of pig delta coronavirus detection, can solve the problems of inaccurate and reliable detection results, aerosol pollution, etc., and achieve the effect of low cost and good specificity

Inactive Publication Date: 2019-04-09
广州动佰生物科技有限公司 +1
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  • Abstract
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Problems solved by technology

LAMP is also one of them, but lamp requires multiple pairs of primers, which is likely to cause aerosol pollution and make the detection results inaccurate and reliable

Method used

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  • PDCoV detection method based on real-time fluorescence RT (reverse transcription) RAA (recombinase-aid amplification) technology
  • PDCoV detection method based on real-time fluorescence RT (reverse transcription) RAA (recombinase-aid amplification) technology
  • PDCoV detection method based on real-time fluorescence RT (reverse transcription) RAA (recombinase-aid amplification) technology

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Embodiment Construction

[0038] The present invention will be described in detail below in conjunction with the accompanying drawings and specific embodiments, and the schematic embodiments and descriptions of the present invention are used to explain the present invention, but are not intended to limit the present invention.

[0039] 1 Materials and methods

[0040] 1.1 Preparation of dsRNA positive standard

[0041] Use TGuide Total Nucleic Acid Extraction Viral DNA / RNA Kit and Automatic Nucleic Acid Extractor to extract PDCoV virus (Genebank No.KY 363868.1) RNA from pig testicular cells cultured and isolated in our laboratory as template RNA. According to the N gene sequence published by PDCoV in GenBank, design RT-PCR primers:

[0042] Upstream primer: 5`~GCTACTCATCCTCAGTTTCGTG~3`;

[0043] Downstream primer: 5`~ACGCTGCTGATTCCTGCT~3`;

[0044] Using One Step PrimeScript TM RT-PCR Kit (TAKARA Company), the reaction system was established according to the instructions, and the primers were used...

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Abstract

The invention discloses a PDCoV detection method based on a real-time fluorescence RT (reverse transcription) RAA (recombinase-aid amplification) technology. The method comprises experiment steps as follows: extracting RNA of PDCoV virus and designing Real-time RT-RAA primers and probes; establishing a Real-time RT-RAA reaction system, conducting a Real-time RT-RAA reaction on the RNA of the virusand obtaining real-time fluorescence detection results. The method has the advantages as follows: the PDCoV is detected simply and quickly at a low cost, the sensitivity of the Real-time RT-RAA detection method is consistent with that of a qPCR detection method, which is 102 copies/mu L, and the specificity is good.

Description

technical field [0001] The invention relates to the technical field of porcine virus detection, in particular to the detection technology of porcine delta coronavirus (PDCoV). Background technique [0002] Porcine Delta coronavirus (PDCoV) is a newly discovered enveloped single-stranded positive-sense RNA virus in 2009, belonging to the Deltacoronavirus genus. The PDCoV genome is about 25kb long, including 5' and 3' untranslated regions and 7 open reading frames, which encode polymerase protein 1a / 1b, spike protein, small membrane protein, membrane protein, nonstructural protein 6, nucleocapsid Shell protein and non-structural protein 7. In 2012, it was first reported in Hong Kong, China, but it was not isolated from diarrheal piglets and feces of American sows until February 2014. Subsequently, PDCoV was also found in sick pigs with diarrhea symptoms in South Korea, mainland China and Thailand. Then it broke out worldwide, causing huge economic losses to the pig industry....

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6844C12R1/93
CPCC12Q1/6844C12Q1/701C12Q2521/107C12Q2521/507C12Q2531/119C12Q2563/107
Inventor 丛潇王华南杨忠艳杨玉翠
Owner 广州动佰生物科技有限公司
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