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一种用于检测四种猪肠道冠状病毒的多重逆转录聚合酶链反应方法的开发。

Development of a multiplex RT-PCR method for the detection of four porcine enteric coronaviruses.

作者信息

Niu Jia-Wei, Li Jin-Hui, Guan Jin-Lian, Deng Ke-Hui, Wang Xiu-Wu, Li Gen, Zhou Xia, Xu Min-Sheng, Chen Rui-Ai, Zhai Shao-Lun, He Dong-Sheng

机构信息

Key Laboratory of Zoonosis Prevention and Control of Guangdong Province, College of Veterinary Medicine of South China Agricultural University, Guangzhou, China.

Ministry of Agriculture of Rural Affairs, Key Laboratory of Animal Disease Prevention of Guangdong Province, Institute of Animal Health, Guangdong Academy of Agricultural Sciences, Scientific Observation and Experiment Station of Veterinary Drugs and Diagnostic Techniques of Guangdong Province, Guangzhou, China.

出版信息

Front Vet Sci. 2022 Nov 8;9:1033864. doi: 10.3389/fvets.2022.1033864. eCollection 2022.

Abstract

Porcine enteric coronaviruses are pathogens that cause viral diarrhea in pigs and are widely prevalent worldwide. Moreover, studies have shown that some porcine enteric coronaviruses can infect humans and poultry. In order to effectively monitor these viruses, it is necessary to establish a multiple detection method to understand their prevalence and conduct in-depth research. Common porcine enteric coronaviruses include Porcine epidemic diarrhea virus (PEDV), Porcine transmissible gastroenteritis virus (TGEV), Porcine delta coronavirus (PDCoV), and Swine acute diarrhea syndrome coronavirus (SADS-CoV). Pigs infected with these viruses have the common clinical symptoms that are difficult to distinguish. A quadruplex RT-PCR (reverse transcription-polymerase chain reaction) method for the simultaneous detection of PEDV, PDCoV, TGEV and SADS-CoV was developed. Four pairs of specific primers were designed for the PEDV gene, PDCoV gene, TGEV gene and SADS-CoV gene. Multiplex RT-PCR results showed that the target fragments of PDCoV, SADS-CoV, PEDV and TGEV could be amplified by this method. and the specific fragments with sizes of 250 bp, 368 bp, 616 bp and 801 bp were amplified, respectively. This method cannot amplify any fragment of nucleic acids of Seneca Valley virus (SVV), Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) and Atypical Porcine Pestivirus (APPV), and has good specificity. The lowest detection limits of PDCoV, PEDV, TGEV and SADS-CoV were 5.66 × 10 copies/μL, 6.48 × 10 copies/μL, 8.54 × 10 copies/μL and 7.79 × 10 copies/μL, respectively. A total of 94 samples were collected from pig farms were analyzed using this method. There were 15 positive samples for PEDV, 3 positive samples for mixed infection of PEDV and PDCoV, 2 positive samples for mixed infection of PEDV and TGEV, and 1 positive sample for mixed infection of PEDV, TGEV, and PDCoV. Multiplex RT-PCR method could detect four intestinal coronaviruses (PEDV, PDCoV, TGEV, and SADS-CoV) in pigs efficiently, cheaply and accurately, which can be used for clinical large-scale epidemiological investigation and diagnosis.

摘要

猪肠道冠状病毒是引起猪病毒性腹泻的病原体,在全球广泛流行。此外,研究表明,一些猪肠道冠状病毒可感染人类和家禽。为有效监测这些病毒,有必要建立一种多重检测方法,以了解它们的流行情况并进行深入研究。常见的猪肠道冠状病毒包括猪流行性腹泻病毒(PEDV)、猪传染性胃肠炎病毒(TGEV)、猪δ冠状病毒(PDCoV)和猪急性腹泻综合征冠状病毒(SADS-CoV)。感染这些病毒的猪具有难以区分的共同临床症状。开发了一种用于同时检测PEDV、PDCoV、TGEV和SADS-CoV的四重RT-PCR(逆转录-聚合酶链反应)方法。针对PEDV基因、PDCoV基因、TGEV基因和SADS-CoV基因设计了四对特异性引物。多重RT-PCR结果表明,该方法可扩增出PDCoV、SADS-CoV、PEDV和TGEV的目标片段。分别扩增出大小为250 bp、368 bp、616 bp和801 bp的特异性片段。该方法不能扩增塞内卡山谷病毒(SVV)、猪繁殖与呼吸综合征病毒(PRRSV)和非典型猪瘟病毒(APPV)的任何核酸片段,具有良好的特异性。PDCoV、PEDV、TGEV和SADS-CoV的最低检测限分别为5.66×10拷贝/μL、6.48×10拷贝/μL、8.54×10拷贝/μL和7.79×10拷贝/μL。使用该方法对从猪场采集的94份样品进行了分析。PEDV阳性样品15份,PEDV和PDCoV混合感染阳性样品3份,PEDV和TGEV混合感染阳性样品2份,PEDV、TGEV和PDCoV混合感染阳性样品1份。多重RT-PCR方法可高效、廉价、准确地检测猪体内的四种肠道冠状病毒(PEDV、PDCoV、TGEV和SADS-CoV),可用于临床大规模流行病学调查和诊断。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/1670/9679136/0710f674fa2a/fvets-09-1033864-g0001.jpg

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