A multiplex reverse transcription polymerase chain rection (mRT-PCR) was developed for simultaneous detection of four RNA viruses in swine. The conserved target sequences directed to classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine epidemic diarrhea virus (PEDV) and transmissible gastroenteritis coronavirus (TGEV) were selected based on alignments of genomic sequences and then specific primers were designed. The mRT-PCR assay was developed and evaluated for its specificity and sensitivity. The expected product from the single viral template was amplified by mRT-PCR and no spurious PCR amplification occurred from the genomic RNA or DNA of other pathogens. For single virus or different combinations of two viruses the detection limit of mRT-PCR was consistent with a single RT-PCR wtith 1 × 103 copies. For different combinations of the three viruses or four viruses, sensitivity of PEDV detection partially decreased. All of positive clinical specimens by the mRT-PCR were identically confirmed using Taqman RT-qPCR. Therefore, the mRT-PCR is a useful tool for epidemiological studies and laboratory diagnosis of single virus and/or mixed infections in swine.

中文翻译：

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多重RT-PCR分析法，用于快速，同时检测猪中的四种RNA病毒。

开发了一种多重逆转录聚合酶链反应技术（mRT-PCR），用于同时检测猪中的四种RNA病毒。根据基因组序列的比对，选择特异的引物，筛选出针对经典猪瘟病毒（CSFV），猪繁殖与呼吸综合征病毒（PRRSV），猪流行性腹泻病毒（PEDV）和可传播性胃肠炎冠状病毒（TGEV）的保守靶序列。被设计。开发了mRT-PCR分析并评估了其特异性和敏感性。通过mRT-PCR扩增了单个病毒模板的预期产物，并且其他病原体的基因组RNA或DNA均未发生伪PCR扩增。对于单个病毒或两种病毒的不同组合，mRT-PCR的检出限与单个RT-PCR的重量为1×103拷贝一致。对于三种病毒或四种病毒的不同组合，PEDV检测的灵敏度部分降低。使用Taqman RT-qPCR相同地证实了所有通过mRT-PCR阳性的临床标本。因此，mRT-PCR是用于猪的单一病毒和/或混合感染的流行病学研究和实验室诊断的有用工具。