四种常见猪肠道病毒多重RT-PCR检测方法的建立及临床应用

Establishment and clinical application of multiplex RT-PCR assay for four common porcine enteroviruses

摘要:

【背景】猪流行性腹泻病毒(porcine epidemic diarrhea virus，PEDV)、猪传染性胃肠炎病毒(transmissible gastroenteritis virus，TGEV)、猪δ冠状病毒(porcine deltacoronavirus，PDCoV)和猪轮状病毒(porcine rotavirus，PoRV)是当前导致猪群发生病毒性腹泻的4种主要病原，并且常发生混合感染。【目的】建立一种可鉴别诊断这4种腹泻病毒病的检测方法，对于临床诊断具有重要意义。【方法】针对PEDV的M蛋白基因、TGEV的N蛋白基因、PDCoV的N蛋白基因和PoRV的VP7蛋白基因分别设计特异性引物，进而构建相应的重组质粒标准品。通过对PCR反应条件优化，建立可同时检测PEDV、TGEV、PDCoV和PoRV的多重RT-PCR检测方法。随后通过敏感性、特异性和重复性试验对该方法的有效性进行验证。【结果】敏感性试验结果显示，对PEDV-M、TGEV-N、PDCoV-N和PoRV-VP7重组质粒标准品的最低检测下限分别为1.75×10²、1.5×10³、1.6×10²和1.6×10²copies/μL；特异性试验结果显示，仅可检出本研究中的4种靶病毒，而猪群常见病毒CSFV、PRRSV、PCV2和PRV未能检出。重复性试验结果显示，选取10⁸、10⁶和10⁴copies/μL 3个不同浓度的重组质粒作为模板，其他条件不变，分别进行5次重复试验，5次试验均扩增出清晰、均匀的条带。对山东各地区送检的52份临床腹泻样品通过建立的四重RT-PCR方法进行检测，发现PEDV、TGEV、PDCoV和PoRV的阳性率分别为37%(19份)、6%(3份)、10%(5份)和25%(13份)。其中PEDV和PoRV混合感染2份(4%)，PEDV和TGEV混合感染2份(4%)，PEDV和PDCoV混合感染1份(2%)。通过单重RT-PCR对该多重RT-PCR临床样品检测结果进行重复验证，结果显示多重RT-PCR与常规单重RT-PCR结果符合率为100%。最后随机挑选5个检测为阳性的临床样本进行测序验证，结果均为相应病毒的基因片段。【结论】本研究建立了可同时检测PEDV、TGEV、PDCoV和PoRV的四重RT-PCR检测方法，研究结果为临床4种猪腹泻病毒病的鉴别诊断及流行病学调查提供了技术手段。

Abstract:

[Background] Porcine epidemic diarrhea virus (PEDV), porcine transmissible gastroenteritis virus (TGEV), porcine deltacoronavirus (PDCoV), and porcine rotavirus (PoRV) are the four main pathogens that cause viral diarrhea in pigs and often occur in mixed infections. [Objective] To establish a method for simultaneous detection of the four viruses in clinical practice. [Methods] The specific primers were designed for the M protein gene of PEDV, N protein gene of TGEV, N protein gene of PDCoV, and VP7 protein gene of RoRV, and the corresponding recombinant plasmids were constructed. By optimizing the PCR conditions, a multiplex RT-PCR assay for the simultaneous detection of PEDV, TGEV, PDCoV, and PoRV was successfully established. Further, the sensitivity, specificity, and reproducibility of the established method were evaluated. [Results] The sensitivity test showed that the multiplex RT-PCR assay had the lower limits of detection of 1.75×10², 1.5×10³, 1.6×10² and 1.6×10² copies/μL for PEDV-M, TGEV-N, PDCoV-N, and PoRV-VP7 recombinant plasmid standards, respectively. The results of specificity test showed that only the four target viruses in this study could be detected, while other common porcine viruses such as classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus type 2 (PCV2), and porcine pseudorabies virus (PRV) could not be detected. The five replicate tests with 10⁸, 10⁶ and 10⁴ copies/μL of recombinant plasmids as templates and other conditions unchanged all produced clear and uniform bands. The 52 clinical diarrhea samples from various regions in Shandong Province were tested by the established quadruple RT-PCR assay. The results showed that the positive rates of PEDV, TGEV, PDCoV, and PoRV were 37% (19 samples), 6% (3 samples), 10% (5 samples) and 25% (13 samples), respectively. 2 (4%), 2 (4%), and 1 (2%) samples showed mixed infections of PEDV and PoRV, mixed infections of PEDV and TGEV, and mixed infections of PEDV and PDCoV, respectively. Further, the results of the multiplex RT-PCR assay were validated by monoplex RT-PCR, which showed 100% compliance. Finally, five positive clinical samples were randomly selected and sent for sequencing to verify the results, and all of them were the gene fragments of the corresponding viruses. [Conclusion] In this study, a quadruple RT-PCR assay for the simultaneous detection of PEDV, TGEV, PDCoV and PoRV was established, and the results provide a technical tool for the differential diagnosis and epidemiological investigation of four clinical porcine diarrhea virus diseases.