猪流行性腹泻病毒(PEDV),猪deltacoronavirus(PDCoV)和传染性胃肠炎病毒(TGEV)是引起猪腹泻的三种临床上常见的冠状病毒,具有难以区分的临床体征和病理变化。快速,这三种病原体的便携式和可靠的鉴别诊断对于迅速实施适当的控制措施至关重要。在这项研究中,我们开发了一种三重核酸测定法,通过靶向PEDVORF1b基因中最保守的基因组区域,将逆转录重组酶辅助扩增(RT-RAA)与侧流测定法(LFA)相结合,PDCoV和TGEV。三重RT-RAA-LFA测定的整个检测过程包括在42°C下10分钟的核酸扩增和在室温下5分钟的视觉LFA读出。该测定法可以特异性区分PEDV,PDCoV和TGEV与任何其他主要猪病原体无交叉反应。敏感性分析表明,三重RT-RAA-LFA测定法能够检测从至少1×100TCID50PEDV的加标粪便样品中提取的病毒RNA,1×104TCID50PDCoV,每个反应1×102TCID50TGEV,分别。进一步的分析表明,PEDV的三组分RT-RAA-LFA的检测限(LOD)为95%,PDCoV,TGEV为每个反应22、478和205拷贝的ORF1b重组质粒,分别。将三重RT-RAA-LFA的诊断性能与PEDV的诊断性能进行了比较,通过平行测试114个临床直肠拭子样品,PDCoV和TGEV各自的商业实时RT-PCR试剂盒。三联RT-RAA-LFA与PEDV实时RT-PCR试剂盒的总诊断符合率,PDCoV和TGEV为100%,99.1%和99.1%,分别,Kappa值分别为1.00、0.958和0.936。总的来说,RT-RAA-LFA检测是一个强大的工具,便携式,视觉,和PEDV的同步鉴别诊断,PDCoV,和TGEV。
Porcine epidemic diarrhea virus (
PEDV), porcine deltacoronavirus (PDCoV) and transmissible gastroenteritis virus (TGEV) are three clinically common coronaviruses causing diarrhea in pigs, with indistinguishable clinical signs and pathological changes. Rapid, portable and reliable differential diagnosis of these three pathogens is crucial for the prompt implementation of appropriate control measures. In this study, we developed a triplex nucleic acid assay that combines reverse transcription recombinase-aided amplification (RT-RAA) with lateral flow assay (LFA) by targeting the most conserved genomic region in the ORF1b genes of
PEDV, PDCoV and TGEV. The entire detection process of the triplex RT-RAA-LFA assay included 10-min nucleic acid amplification at 42 °C and 5-min visual LFA readout at room temperature. The assay could specifically differentiate
PEDV, PDCoV and TGEV without cross-reaction with any other major swine pathogens. Sensitivity analysis showed that the triplex RT-RAA-LFA assay was able to detect the viral RNA extracted from the spiked fecal samples with the minimum of 1 × 100 TCID50 PEDV, 1 × 104 TCID50 PDCoV, and 1 × 102 TCID50 TGEV per reaction, respectively. Further analysis showed that the 95 % detection limit (LOD) of triplex RT-RAA-LFA for
PEDV, PDCoV, and TGEV were 22, 478, and 205 copies of recombinant plasmids per reaction, respectively. The diagnostic performance of triplex RT-RAA-LFA was compared with that of PEDV, PDCoV and TGEV respective commercial real-time RT-PCR kits by testing 114 clinical rectal swab samples in parallel. The total diagnostic coincidence rates of triplex RT-RAA-LFA with real-time RT-PCR kits of
PEDV, PDCoV and TGEV were 100 %, 99.1 % and 99.1 %, respectively, and their Kappa values were 1.00, 0.958 and 0.936, respectively. Collectively, the RT-RAA-LFA assay is a powerful tool for the rapid, portable, visual, and synchronous differential diagnosis of PEDV, PDCoV, and TGEV.