Areekit Supatra, Tangjitrungrot Pongbun, Khuchareontaworn Sintawee, Rattanathanawan Kankanit, Jaratsing Pornpun, Yasawong Montri, Chansiri Gaysorn, Viseshakul Nareerat, Chansiri Kosum
Innovative Learning Center, Srinakharinwirot University, Bangkok 10110, Thailand.
Center of Excellence in Biosensors, Srinakharinwirot University, Bangkok 10110, Thailand.
Curr Issues Mol Biol. 2022 Nov 3;44(11):5427-5439. doi: 10.3390/cimb44110368.
Porcine epidemic diarrhea virus (PEDV) and porcine circovirus type 2 (PCV2) are both important global pathogenic viruses which have a significant impact on the swine industry. In this study, a duplex loop-mediated isothermal amplification (duplex LAMP) method was developed in combination with lateral flow dipstick (LFD) for simultaneous detection of PEDV and PCV2 using specific sets of primers and probes designed based on the conserved regions of a spike gene (KF272920) and an ORF gene (EF493839), respectively. The limit of detection (LOD) values of the duplex LAMP-LFD for the detection of PEDV and PCV2 were 0.1 ng/µL and 0.246 ng/µL, respectively. The LOD of duplex LAMP-LFD was 10-times more sensitive than conventional PCR and RT-PCR-agarose gel-electrophoresis (PCR-AGE and RT-PCR-AGE). No cross-reaction to each other and to other pathogenic viruses that can infect pigs were observed according to analytical specificity tests. The duplex LAMP-LFD method for the simultaneous detection of PEDV and PCV2 co-infection could be completed within approximately 1.5 h, and only a simple heating block was required for isothermal amplification. The preliminary validation using 50 swine clinical samples with positive and negative PEDV and/or PCV2 revealed that the sensitivity, specificity, and accuracy of duplex LAMP-LFD were all 100% in comparison to conventional PCR and RT-PCR. Hence, this study suggests that duplex LAMP-LFD is a promising tool for the early detection and initial screening of PEDV and PCV2, which could be beneficial for prevention, planning, and epidemiological surveys of these diseases.
猪流行性腹泻病毒（PEDV）和猪圆环病毒2型（PCV2）都是对全球养猪业有重大影响的重要致病病毒。在本研究中，结合侧向流动试纸条（LFD）开发了一种双重环介导等温扩增（duplex LAMP）方法，分别使用基于刺突基因（KF272920）和开放阅读框基因（EF493839）保守区域设计的特定引物和探针组，同时检测PEDV和PCV2。双重LAMP-LFD检测PEDV和PCV2的检测限（LOD）值分别为0.1 ng/µL和0.246 ng/µL。双重LAMP-LFD的LOD比传统PCR和RT-PCR-琼脂糖凝胶电泳（PCR-AGE和RT-PCR-AGE）灵敏10倍。根据分析特异性试验，未观察到彼此之间以及与其他可感染猪的致病病毒的交叉反应。用于同时检测PEDV和PCV2共感染的双重LAMP-LFD方法可在约1.5小时内完成，等温扩增仅需一个简单的加热块。使用50份PEDV和/或PCV2阳性和阴性猪临床样本进行的初步验证表明，与传统PCR和RT-PCR相比，双重LAMP-LFD的敏感性、特异性和准确性均为100%。因此，本研究表明双重LAMP-LFD是一种用于PEDV和PCV2早期检测和初步筛查的有前景的工具，这可能有利于这些疾病的预防、规划和流行病学调查。
