Multiplex RT-PCR detection method of porcine delta coronavirus, porcine epidemic diarrhea virus and porcine sapelovirus, and applications thereof

A technology of porcine epidemic diarrhea and RT-PCR, which is applied in the direction of microorganism-based methods, biochemical equipment and methods, and microorganism measurement/inspection, to achieve the effects of improving amplification efficiency, high pathogen sensitivity rate, and strong specificity

Inactive Publication Date: 2020-03-24
HENAN AGRICULTURAL UNIVERSITY
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Problems solved by technology

[0005] The present invention proposes a multiplex RT-PCR detection method and application of porcine delta coronavirus, porcine epidemic diarrhea virus and porcine sapero virus...

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  • Multiplex RT-PCR detection method of porcine delta coronavirus, porcine epidemic diarrhea virus and porcine sapelovirus, and applications thereof
  • Multiplex RT-PCR detection method of porcine delta coronavirus, porcine epidemic diarrhea virus and porcine sapelovirus, and applications thereof
  • Multiplex RT-PCR detection method of porcine delta coronavirus, porcine epidemic diarrhea virus and porcine sapelovirus, and applications thereof

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Embodiment

[0029] 1. Experimental materials

[0030] Porcine delta coronavirus (PDCoV, CH-01 strain), porcine Sapero virus (PSV) positive disease material, porcine epidemic diarrhea virus (PEDV) positive disease material, porcine pseudorabies virus (PRV), porcine Porcine Bocavirus (PBoV), swine fever virus (Classical swine fever virus, CSFV) and Porcine circovirus (Porcine circovirus, PCV) positive disease materials were all kept by the Henan Provincial Key Laboratory of Animal Food Safety. Porcine respiratory and reproductive syndrome virus (RRRSV) vaccine was purchased from Harbin Veken Biotechnology Company. The disease materials were obtained from the small intestines of piglets that died of diarrhea in some cities in Henan Province from 2017 to 2019 and 92 feces of piglets with diarrhea.

[0031] 2. Main reagents

[0032] Reverse transcription kits were purchased from Nanjing Novizan Biotechnology Co., Ltd. Biotechnology Company; DNA-Marker, pMD18-T vector, competent cell DH5α, PC...

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Abstract

The invention belongs to the field of virus detection, and particularly relates to a multiplex RT-PCR detection method of porcine delta coronavirus, porcine epidemic diarrhea virus and porcine sapelovirus, and applications thereof. Three pairs of specific primers for synthesizing conserved sequences of PEDV M gene, PDCoV N gene and PSV 2C gene are designed; then, RT-PCR reaction conditions are optimized, the multiplex RT-PCR diagnosis method capable of simultaneously detecting porcine delta coronavirus, porcine epidemic diarrhea virus and porcine sapelovirus is established, and specificity, sensitivity and repeatability analysis is performed on the established method. It is shown by results the method disclosed by the invention has very good specificity and relatively high sensitivity; theminimum detection limit of PDCoV is 140 copies/<mu>L, the minimum detection limit of PEDV is 153 copies/<mu>L, and the minimum detection limit of PSV is 1570 copies/<mu>L.

Description

technical field [0001] The invention belongs to the field of virus detection, in particular to a multiplex RT-PCR detection method and application of porcine delta coronavirus, porcine epidemic diarrhea virus and porcine Sapero virus. Background technique [0002] In recent years, swine viral diseases have caused serious harm to the global swine industry. The occurrence and prevalence of diseases have become increasingly serious, the incidence rate has increased year by year, and the mixed infection and secondary infection of multiple pathogens have become more and more common. Porcine deltacoronavirus (PDCoV), porcine epidemic diarrhea virus (PEDV) and porcine Sapero virus (PSV) can all cause similar clinical symptoms such as severe dehydration and diarrhea in piglets, and the clinical mixed infection phenomenon is compared It is common, which brings many difficulties to the diagnosis and prevention and control of the disease. Although traditional animal virus disease diag...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/686C12R1/94
CPCC12Q1/686C12Q1/701C12Q2521/107C12Q2537/143
Inventor 胡慧魏战勇丁庆文张红垒郑兰兰夏璐张云飞李倩李林姣单法
Owner HENAN AGRICULTURAL UNIVERSITY
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