Abstract
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.
Highlights
Influenza A virus, including swine influenza and avian influenza, is a highly contagious virus that causes annual epidemic and sometimes pandemic disease in humans [1]
Construction of the LabChip real-time polymerase chain reaction (PCR) system In our study, polymer chips were fabricated for such reasons as low cost, light weight, and excellent processing ability
We suggest that the ultra-fast LabChip real-time PCR system (G2–3) provides a faster and more accurate system for diagnosing various infectious diseases (Figure 7)
Summary
Influenza A virus, including swine influenza and avian influenza, is a highly contagious virus that causes annual epidemic and sometimes pandemic disease in humans [1]. Many real-time PCR-based assays have been developed for detecting individual subtypes or specific pandemic influenza strains [17]–[23]. Kawai et al reported a novel detection method, called the RT-SmartAmp assay, for one-step detection of the pandemic influenza strain A/H1N1 of 2009 [24] Using this PCR system, the reaction time was reduced to 40 min. Fluidic chip-based real-time PCR has received attention for its ability to achieve rapid molecular quantitative analysis of infectious disease pathogens due to its excellent sensitivity and specificity [29]–[32]. We developed and validated a fluidic chiptype real-time PCR system It achieved rapid quantitative analysis (15 min to perform 30 cycles of real-time PCR). Using clinical samples of influenza A/H1N1, we established that the fluidic chip-based real-time PCR system had excellent sensitivity and specificity, which were nearly identical to those of a conventional tube-type real-time PCR system
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