Kumar Jyoti S, Yadav Pragya D, Shete Anita M, Majumdar Triparna, Patil Savita, Dash Paban Kumar
Division of Virology, Defence Research and Development Establishment, Gwalior 474002, India.
Indian Council of Medical Research, National Institute of Virology, Pune, 411021, India.
Int J Infect Dis. 2021 Nov;112:346-351. doi: 10.1016/j.ijid.2021.01.041. Epub 2021 Jan 21.
Kyasanur forest disease (KFD), a re-emerging tick-borne viral disease, causes severe hemorrhagic fever in humans and nonhuman primates. KFD virus (KFDV) is a member of the genus Flavivirus. KFD is now increasingly reported outside its endemic zone in India. Rapid and specific detection of the KFDV plays a critical role in containment of the outbreak. The diagnosis of KFD currently relies on real-time RT-PCR, nested RT-PCR, end point RT-PCR, and serodiagnostic assay. These assays are tedious, time-consuming, and cannot be used as a routine screening platform.
The present study was aimed to develop a one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for molecular diagnosis of KFD.
The gene amplification reaction was accomplished by incubation at a constant temperature of 63°C for 60min.
The limit of detection of RT-LAMP assay was 10 copies. KFD RT-LAMP assay was successfully evaluated with diverse host samples including humans, monkeys, and tick. The assay correctly picked up different KFD isolates indicating its applicability for divergent strains. Comparative evaluation of RT-LAMP assay with quantitative TaqMan real-time RT-PCR revealed 100% concordance. No cross-reaction with related flavi and other hemorrhagic fever viruses was observed, indicating its high specificity.
The RT-LAMP test developed in this study will serve as a rapid, sensitive alternate detection method for KFDV infection and would be useful for high throughput screening of clinical samples in resource limited areas during outbreaks.
基孔肯雅森林病（KFD）是一种再度出现的蜱传病毒性疾病，可导致人类和非人类灵长类动物出现严重出血热。KFD病毒（KFDV）是黄病毒属的成员。目前，在印度KFD流行区以外越来越多地报告了该疾病。快速、特异性地检测KFDV在控制疫情爆发中起着关键作用。KFD的诊断目前依赖于实时逆转录聚合酶链反应（RT-PCR）、巢式RT-PCR、终点RT-PCR和血清诊断检测。这些检测方法繁琐、耗时，且不能用作常规筛查平台。
本研究旨在开发一种用于KFD分子诊断的一步法逆转录环介导等温扩增（RT-LAMP）检测方法。
基因扩增反应通过在63°C恒温下孵育60分钟完成。
RT-LAMP检测的检测限为10个拷贝。KFD RT-LAMP检测已成功用于包括人类、猴子和蜱在内的多种宿主样本的评估。该检测方法能够正确识别不同的KFD分离株，表明其适用于不同的菌株。RT-LAMP检测与定量TaqMan实时RT-PCR的比较评估显示一致性为100%。未观察到与相关黄病毒和其他出血热病毒的交叉反应，表明其具有高度特异性。
本研究开发的RT-LAMP检测将作为一种快速、灵敏的KFDV感染替代检测方法，在疫情爆发期间，对于资源有限地区临床样本的高通量筛查将非常有用。
