Center for Dengue Fever Control and Research, Kaohsiung Medical University, Kaohsiung, Taiwan.
School of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan.
PLoS One. 2019 Jul 10;14(7):e0218139. doi: 10.1371/journal.pone.0218139. eCollection 2019.
The insulated isothermal PCR (iiPCR) technology enables consistent PCR amplification and detection in a simple heating device. A pan-dengue virus (DENV) RT-iiPCR, targeting the 5' untranslated region, was validated previously on the semi-automated POCKIT combo system (involving separate devices for nucleic acid extraction and PCR amplification/detection) to offer performance comparable to a laboratory real-time PCR. Working on the same technologies, a compact automated sample-in-answer-out system (POCKIT Central Nucleic Acid Analyser) has been available commercially for iiPCR, minimizing human error risks and allowing easy molecular bio-detection near points of need. Here, we evaluated the analytical and clinical performance of the pan-DENV RT-iiPCR on the fully automated system by comparison to those on the semi-automated system.
METHODOLOGY/PRINCIPAL FINDINGS: Testing sera containing serial diluted DENV-1, -2, -3, or -4 cell culture stock, the pan-DENV RT-iiPCR system had similar 100% detection endpoints on the two systems; i.e. at 1, 10, 1 and 10 PFU/ml, respectively, on the fully automated system, and at 10, 1, 10 and 10 PFU/ml, respectively, on the semi-automated system. Furthermore, both fully automated and semi-automated PCR system can detect all four DENV serotypes in mosquitos. Clinical performance of the reagent on the two systems was evaluated by testing 60 human serum samples. Both systems detected the same 40 samples (ten DENV-1, -2, -3, and -4 positive each) and did not detect the other 20; 100% agreement (κ = 1) was found between the two systems.
CONCLUSIONS/SIGNIFICANCE: With performance comparable to a previously validated system, the fully-automated PCR system allows applications of the pan-DENV reagent as a useful tool near points of need to facilitate easy, fast and effective detection of dengue virus and help mitigate versatile public health challenges in the control and management of dengue disease.
隔热等温 PCR（iiPCR）技术可在简单的加热装置中实现一致的 PCR 扩增和检测。先前在半自动 POCKIT combo 系统（涉及核酸提取和 PCR 扩增/检测的独立设备）上验证了针对 5'非翻译区的泛登革热病毒（DENV）RT-iiPCR，其性能可与实验室实时 PCR 相媲美。基于相同的技术，一种紧凑的自动化样本进结果出系统（POCKIT Central Nucleic Acid Analyser）已可用于 iiPCR，可最大程度降低人为错误风险，并允许在需要的地方进行简便的分子生物检测。在这里，我们通过与半自动系统比较，评估了全自动系统上的泛 DENV RT-iiPCR 的分析和临床性能。
方法/主要发现：用含有系列稀释的 DENV-1、-2、-3 或-4 细胞培养物库存的血清进行测试，pan-DENV RT-iiPCR 系统在两个系统上具有相似的 100%检测终点；即全自动系统上分别为 1、10、1 和 10 PFU/ml，半自动系统上分别为 10、1、10 和 10 PFU/ml。此外，全自动和半自动 PCR 系统都可以检测到蚊子中的所有四种 DENV 血清型。通过测试 60 份人血清样本评估了试剂在两个系统上的临床性能。两个系统都检测到了相同的 40 个样本（每个样本分别为 10 个 DENV-1、-2、-3 和-4 阳性），未检测到另外 20 个样本；两个系统之间发现了 100%的一致性（κ=1）。
结论/意义：与先前验证的系统性能相当，全自动 PCR 系统允许将泛 DENV 试剂作为一种有用的工具在需要的地方应用，以方便、快速和有效地检测登革热病毒，并有助于减轻控制和管理登革热疾病方面的多种公共卫生挑战。
