Virology Division, Defence Research Development Establishment, Jhansi Road, Gwalior, 474002, India.
Virology Division, Defence Research Development Establishment, Jhansi Road, Gwalior, 474002, India.
Virus Res. 2023 Oct 2;335:199180. doi: 10.1016/j.virusres.2023.199180. Epub 2023 Aug 1.
Among recently prevalent tick-borne infections in India, Kyasanur Forest Virus Disease (KFD) is an important public health concern. During last decade the emergence of cases apart from endemic zone raised concern about case positivity. Early diagnosis is therefore very important in disease management and primary containment. This study, aimed to develop a simplified viral RNA extraction in combination to dry down format of novel isothermal assay for (Reverse Transcription- Polymerase Spiral reaction) specific and rapid identification of Kyasanur Forest Disease Virus targeting viral envelope gene. The one step method was optimized by magnetic bead based viral RNA extraction followed by isothermal RT-PSR assay in heat bath at 63⁰C for 60 minutes. Further, visual results interpretation was done by color change of Hydroxy Naphthol Blue dye. The detection limit of the assay was found 10 RNA copies/rxn with comparable to silica column based viral RNA combined real time qPCR. No cross reactivity was observed with other closely related flaviviruses. The assay was evaluated with clinical samples has shown >99% concordance between two methods. This is the first report of sample extraction coupled isothermal detection of KFD in a simplified manner without a need of any hi-end equipment. The assay developed here has potential to use as an alternate for field-based detection in resource limited settings for KFD.
在印度最近流行的蜱传感染中，Kyasanur 森林病毒病 (KFD) 是一个重要的公共卫生关注点。在过去的十年中，除了流行地区之外，病例的出现引起了人们对病例阳性率的关注。因此，早期诊断在疾病管理和初步控制中非常重要。本研究旨在开发一种简化的病毒 RNA 提取方法，并结合新型等温测定法的干样格式，用于（逆转录-聚合酶螺旋反应）特异性和快速鉴定 Kyasanur 森林病毒，该方法针对病毒包膜基因。通过基于磁珠的病毒 RNA 提取，一步法进行了优化，然后在 63°C 的水浴中进行等温 RT-PSR 检测 60 分钟。进一步通过羟萘酚蓝染料的颜色变化进行直观的结果解释。该测定的检测限为 10 RNA 拷贝/反应，与基于硅胶柱的病毒 RNA 实时 qPCR 相当。与其他密切相关的黄病毒没有交叉反应。该测定方法用临床样本进行评估，两种方法之间的一致性>99%。这是首次以简化的方式报告了 KFD 的样品提取和等温检测，而无需任何高端设备。该方法具有在资源有限的环境中用于现场检测的潜力。
