Polymeric LabChip real-time PCR as a point-of-care-potential diagnostic tool for rapid detection of influenza A/H1N1 virus in human clinical specimens

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.

Figure 1. Construction of the plastic LabChip.

The schematic diagram (A) and actual image (B) of the fluidic flow chip.

Figure 2. Thirty cycles of the ultra-fast LabChip real-time PCR system.

The PCR was completed in 15 min within a 0.5°C temperature variation. A. View of NBS UltraFast LabChip Real-time PCR G2–3. B. Plot of 30 thermocycles over 900 s (15 min). C. Close-up view of 72°C, ranging from 72.08°C to 72.40°C, indicating less than 0.5-degree variation.

Figure 3. Amplification efficiency in the plastic chip.

A positive control cDNA was amplified by tube-type and fluidic chip-type PCR. The electrophoresed amplicons are shown in an agarose gel.

Figure 4. Detection sensitivity of LabChip real-time PCR.

LabChip real-time PCR was compared to conventional tube-type real-time PCR for its sensitivity to detect positive template DNA. The Ct values obtained from the LabChip real-time PCR were comparable to those from tube-type real-time PCR (Roche LightCycler 1.5).

Figure 5. LOD (Limit of detection) of LabChip real-time PCR.

Serial dilution of in vitro transcribed RNA was tested using LabChip real-time PCR and tube-type real-time PCR (Bio-Rad CFX96). The Ct values obtained from both PCR systems were compared in a table. Test was performed in duplicate. PCR amplicons were confirmed by gel electrophoresis. NTC, no template control.

Figure 6. Clinical evaluation of LabChip real-time PCR.

Clinical swab samples were used to evaluate the LabChip real-time PCR. The results were compared to those from tube-type real-time PCR (Bio-Rad CFX96). Resultant examples of both real-time PCR assays are shown. PCR amplicons were further analyzed by gel electrophoresis. PC, positive control; NC, no template control.

Figure 7. Flowchart of the ultra-fast LapChip real-time PCR to detect influenza A virus.

Viral RNA is isolated from clinical samples and reverse transcription is performed to generate cDNA. SYBR green real-time PCR is conducted using synthesized cDNA in a LabChip. Ultra-fast real-time PCR is used, and the results are obtained within 15 min.