Microdroplet Sandwich Real-Time RT-PCR for Detection of Pandemic and Seasonal Influenza Subtypes

As demonstrated by the recent 2012/2013 flu epidemic, the continual emergence of new viral strains highlights the need for accurate medical diagnostics in multiple community settings. If rapid, robust, and sensitive diagnostics for influenza subtyping were available, it would help identify epidemics, facilitate appropriate antiviral usage, decrease inappropriate antibiotic usage, and eliminate the extra cost of unnecessary laboratory testing and treatment. Here, we describe a droplet sandwich platform that can detect influenza subtypes using real-time reverse-transcription polymerase chain reaction (rtRT-PCR). Using clinical samples collected during the 2010/11 season, we effectively differentiate between H1N1p (swine pandemic), H1N1s (seasonal), and H3N2 with an overall assay sensitivity was 96%, with 100% specificity for each subtype. Additionally, we demonstrate the ability to detect viral loads as low as 104 copies/mL, which is two orders of magnitude lower than viral loads in typical infected patients. This platform performs diagnostics in a miniaturized format without sacrificing any sensitivity, and can thus be easily developed into devices which are ideal for small clinics and pharmacies.

正如2012/2013年暴发的流感疫情所证实的那样，新型病毒毒株的持续出现凸显了在多类社区场景中开展精准医学诊断的迫切需求。若能拥有快速、稳定且灵敏度优异的流感分型诊断技术，将有助于疫情识别、推动合理使用抗病毒药物、减少不当抗生素使用，并消除不必要实验室检测与治疗带来的额外成本。
本研究中，我们介绍了一种液滴夹心平台，可借助实时逆转录聚合酶链式反应（real-time reverse-transcription polymerase chain reaction，rtRT-PCR）检测流感病毒亚型。我们利用2010/11流感季采集的临床样本，成功区分了H1N1p（猪源性大流行株）、H1N1s（季节性流感株）及H3N2亚型，整体检测灵敏度达96%，各亚型的特异性均为100%。
此外，本平台可检测低至10^4 拷贝/毫升的病毒载量，比典型感染患者体内的病毒载量低两个数量级。
该平台采用微型化设计开展诊断检测，且未牺牲任何检测灵敏度，因此可轻松开发为适用于小型诊所与药房的理想设备。