Zoonosis Research Center, Department of Infection Biology, Wonkwang University School of Medicine, Iksan, Jeonbuk, Republic of Korea.
PLoS One. 2012;7(12):e53325. doi: 10.1371/journal.pone.0053325. Epub 2012 Dec 28.
It is clinically important to be able to detect influenza A/H1N1 virus using a fast, portable, and accurate system that has high specificity and sensitivity. To achieve this goal, it is necessary to develop a highly specific primer set that recognizes only influenza A viral genes and a rapid real-time PCR system that can detect even a single copy of the viral gene. In this study, we developed and validated a novel fluidic chip-type real-time PCR (LabChip real-time PCR) system that is sensitive and specific for the detection of influenza A/H1N1, including the pandemic influenza strain A/H1N1 of 2009. This LabChip real-time PCR system has several remarkable features: (1) It allows rapid quantitative analysis, requiring only 15 min to perform 30 cycles of real-time PCR. (2) It is portable, with a weight of only 5.5 kg. (3) The reaction cost is low, since it uses disposable plastic chips. (4) Its high efficiency is equivalent to that of commercially available tube-type real-time PCR systems. The developed disposable LabChip is an economic, heat-transferable, light-transparent, and easy-to-fabricate polymeric chip compared to conventional silicon- or glass-based labchip. In addition, our LabChip has large surface-to-volume ratios in micro channels that are required for overcoming time consumed for temperature control during real-time PCR. The efficiency of the LabChip real-time PCR system was confirmed using novel primer sets specifically targeted to the hemagglutinin (HA) gene of influenza A/H1N1 and clinical specimens. Eighty-five human clinical swab samples were tested using the LabChip real-time PCR. The results demonstrated 100% sensitivity and specificity, showing 72 positive and 13 negative cases. These results were identical to those from a tube-type real-time PCR system. This indicates that the novel LabChip real-time PCR may be an ultra-fast, quantitative, point-of-care-potential diagnostic tool for influenza A/H1N1 with a high sensitivity and specificity.
临床需要一种快速、便携且准确的系统来检测甲型流感病毒，这种系统具有高特异性和高灵敏度。为了实现这一目标，需要开发一种高特异性的引物组，该引物组只能识别甲型流感病毒基因，以及一种快速实时 PCR 系统，该系统甚至可以检测到单个病毒基因的拷贝。在这项研究中，我们开发并验证了一种新型的流体芯片式实时 PCR（LabChip 实时 PCR）系统，该系统对甲型流感病毒，包括 2009 年的大流行流感病毒 A/H1N1 株具有高度的特异性和灵敏度。这个 LabChip 实时 PCR 系统具有以下几个显著特点：（1）它可以快速进行定量分析，仅需 15 分钟即可完成 30 个循环的实时 PCR。（2）它便于携带，重量仅为 5.5 公斤。（3）反应成本低，因为它使用一次性塑料芯片。（4）它的效率高，与市售的管型实时 PCR 系统相当。与传统的硅基或玻璃基 labchip 相比，开发的一次性 LabChip 是一种经济、可热传递、透光且易于制造的聚合物芯片。此外，我们的 LabChip 在微通道中具有较大的表面积与体积比，这对于克服实时 PCR 过程中温度控制所需的时间非常重要。使用针对甲型流感病毒 A/H1N1 的血凝素（HA）基因的新型引物组和临床标本对 LabChip 实时 PCR 系统的效率进行了验证。使用 LabChip 实时 PCR 对 85 个人类临床拭子样本进行了测试。结果显示，该系统的灵敏度和特异性均为 100%，检测出 72 例阳性和 13 例阴性病例。这些结果与管型实时 PCR 系统的结果完全一致。这表明，新型 LabChip 实时 PCR 可能是一种超快速、定量、有即时诊断潜力的流感 A/H1N1 诊断工具，具有高灵敏度和特异性。
