猪丁型冠状病毒与猪流行性腹泻病毒双重实时荧光定量RT-PCR方法的建立和初步应用

doi:10.11843/j.issn.0366-6964.2018.04.025

Abstract

Abstract:

Porcine deltacoronavirus (PDCoV) and porcine epidemic diarrhea virus (PEDV) are enterpathogenic coronavirus which cause acute diarrhea, vomiting,dehydration and mortality in neonatal piglets. In order to establish a real-time reverse transcription quantitative PCR (RT-qPCR) assay to detect PDCoV and PEDV, two pairs of specific primers were designed according to the conservative sequence of N gene (PDCoV) and M gene (PEDV) registered in GenBank. The genes of the conservative region were amplified and cloned into pMD19-T vector. Taking Mix 10 times the gradient dilution of recombinant plasmid as a standard template, a RT-qPCR to PDCoV and PEDV was established. The sensitivity, specificity and repeatability were tested. Results showed that the sensitivity of this RT-qPCR assay was 51 and 32 copies·μL^-1 for PDCoV and PEDV, respectively. No cross-reaction was detected to PBoV, TGEV, PRV and PCV2. The RT-qPCR assay was applied to the detection of 130 clinical samples collected from Hebei province during 2016-2017. PDCoV positive rate was 16.9%, PEDV positive rate was 66.2%, and co-infection was 2.3%. The sensitivity of fluorescence quantitative PCR was significantly higher than that of ordinary RT-PCR. These results indicated that the detection assay could be applied for rapid and high through-put diagnosis of PDCoV and PEDV.

Key words: porcine deltacoronavirus, porcine epidemic diarrhea virus, real-time quantitative PCR, clinical detection

CLC Number:

LUO Shang-xing, FAN Jing-hui, LIU Bao-jing, SHI Qian-kai, HOU Lin-shan, ZUO Yu-zhu. Establishment and Application of the Real-time Reverse Transcription Quantitative PCR Assay for Porcine Epidemic Diarrhea Virus and Porcine Deltacoronavirus[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2018, 49(4): 852-858.

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Establishment and Application of the Real-time Reverse Transcription Quantitative PCR Assay for Porcine Epidemic Diarrhea Virus and Porcine Deltacoronavirus

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