建立一种三重 RT-RAA-LFA 检测方法，用于快速鉴别诊断猪流行性腹泻病毒、猪德尔塔冠状病毒和传染性胃肠炎病毒。
Development of a triplex RT-RAA-LFA assay for the rapid differential diagnosis of porcine epidemic diarrhea virus, porcine deltacoronavirus and transmissible gastroenteritis virus.
机构信息
National Key Laboratory of Veterinary Public Health and Safety, College of Veterinary Medicine, China Agricultural University, Beijing, 100193, China; Key Laboratory of Animal Epidemiology of Ministry of Agriculture and Rural Affairs, College of Veterinary Medicine, China Agricultural University, Beijing, 100193, China.
National Key Laboratory of Veterinary Public Health and Safety, College of Veterinary Medicine, China Agricultural University, Beijing, 100193, China; Beijing Key Laboratory of Detection Technology for Animal Derived Food Safety, Beijing Laboratory for Food Quality and Safety, College of Veterinary Medicine, China Agricultural University, Beijing, 100193, China.
出版信息
Microb Pathog. 2024 Oct;195:106885. doi: 10.1016/j.micpath.2024.106885. Epub 2024 Aug 24.
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 × 10 TCID PEDV, 1 × 10 TCID PDCoV, and 1 × 10 TCID 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.
猪流行性腹泻病毒（PEDV）、猪德尔塔冠状病毒（PDCoV）和传染性胃肠炎病毒（TGEV）是三种临床上常见的冠状病毒，可引起猪腹泻，其临床症状和病理变化难以区分。快速、便携且可靠的鉴别诊断这三种病原体对于及时采取适当的控制措施至关重要。在本研究中，我们针对 PEDV、PDCoV 和 TGEV 的 ORF1b 基因最保守的基因组区域，开发了一种三重核酸检测方法，该方法结合了逆转录重组酶辅助扩增（RT-RAA）和侧向流动分析（LFA）。三重 RT-RAA-LFA 检测法的整个检测过程包括在 42°C 下进行 10 分钟的核酸扩增和在室温下进行 5 分钟的可视 LFA 读数。该检测法能够特异性地区分 PEDV、PDCoV 和 TGEV，与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 检测法能够检测到每反应中最低浓度为 1×10 TCID PEDV、1×10 TCID PDCoV 和 1×10 TCID TGEV 的病毒 RNA。进一步分析表明，三重 RT-RAA-LFA 对 PEDV、PDCoV 和 TGEV 的 95%检测限（LOD）分别为 22、478 和 205 个重组质粒拷贝/反应。通过对 114 份临床直肠拭子样本进行平行检测，比较了三重 RT-RAA-LFA 与 PEDV、PDCoV 和 TGEV 各自的商业实时 RT-PCR 试剂盒的诊断性能。三重 RT-RAA-LFA 与 PEDV、PDCoV 和 TGEV 实时 RT-PCR 试剂盒的总诊断符合率分别为 100%、99.1%和 99.1%，Kappa 值分别为 1.00、0.958 和 0.936。综上所述，RT-RAA-LFA 检测法是一种快速、便携、可视化、同步鉴别诊断 PEDV、PDCoV 和 TGEV 的有力工具。
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