Volume 1 Issue 5 - September 21, 2007
Integrated Reverse Transcription Polymerase Chain Reaction Systems for Virus Detection
Kang-Yi Lien1, Wan-Chi Lee2, Huan-Yao Lei3 and Gwo-Bin Lee1,2*
  • Institute of Nanotechnology and Microsystems Engineering, National Cheng Kung University, Tainan Taiwan 701
  • Department of Engineering Science, National Cheng Kung University, Tainan, Taiwan 701
  • Department of Microbiology and Immunology, National Cheng Kung University, Tainan, Taiwan 701
*E-mail: gwobin@mail.ncku.edu.tw
Paper published in Biosensors and Bioelectronics, vol. 22, pp. 1739-1748, 2007.
Figure 1
This study reports on an integrated micro reverse transcription polymerase chain reaction (RT-PCR) system for molecular diagnosis of microorganisms automatically. The experimental process of the integrated micro RT-PCR diagnosis system is shown in Figure 1. The magnetic beads have been conjugated with a specific antibody of the interested target antigen (Fig. 1a), followed by mixing viruses with the beads (Fig. 1b). The antibodies on the beads with a high selectivity will bind to the specific antigens on the target virus so that the viruses will adhere to the surface of the magnetic beads (Fig. 1c). In order to wash out the unstable substances in the biological medium which may easily interfere with RNA amplification, the microcoils were turned on to attract the magnetic beads while the fluids still flew through the pretreatment chamber (Fig. 1d). The washing process can be completed in five minutes to utilize the micropumps continuously and the purification of the virus sample was completed by the specific antibody conjugated on the magnetic bead. Then the magnetic beads with target viruses were transported into the subsequent PR-PCR chamber while the magnetic field was turned off (Fig. 1e). Meanwhile, the RT-PCR reagents were pumped into the detection chamber to perform cell lysis, reverse transcription, and polymerase chain reaction (Fig. 1f-1g).

Nucleic acid amplification techniques, including PCR and RT-PCR, are popular methods used for many applications including genetic identification and disease diagnosis of a DNA or RNA molecule. PCR is a popular procedure in molecular biology for generic analysis. During the PCR procedure, the concentration of a certain segment of double-stranded DNA is doubled through a thermal cycling process involving three different temperatures. Typically, PCR utilizes temperatures in the ranges of 90-95°C for denaturation of the double-stranded DNA, 50-65°C for the hybridization of the primers, and 70-75°C for DNA extension. In vitro DNA amplification using PCR provides a rapid and sensitive means of detecting pathogens in clinical specimens, and hence has considerable implications for the diagnostic microbiology field. Similarly, RNA is employed as the template to synthesize a complimentary DNA (cDNA) using a reverse transcriptase process during the RT-PCR process.

Figure 2
To realize the experimental process described above, Figure 2 shows the photograph of the integrated RT-PCR system. The integrated microsystem comprises three major components, a purification module, a microfluidic module, and a micro temperature control module. The purification module consisting of the microcoils can perform collection and enrichment of virus samples by capturing specific target viruses on the superparamagnetic beads, and then collecting these beads by microcoils within the pretreatment chamber. The microfluidic module can perform the mixing, incubation and transportation of the bio-samples in the microchannels which connected to all chambers by utilizing the novel pneumatic micropump. The pneumatic micropump comprised of three individual polydimethylsiloxane (PDMS) membranes that can be driven by only one electromagnetic valve. The compressed air fills up the cavities formed from PDMS membranes subsequentially such that the solutions can be pushed forward in a specific direction. The micro temperature control module, which is made of two micro heaters, a micro temperature sensor and an application specific integrated circuit (ASIC) controller, can perform the cell lysis and the RT-PCR process in an automatic fashion.

Figure 3
This study successfully performs the specific detection of two different types of viruses, Dengue virus serotype 2 and Enterovirus 71 (EV 71) using this developed integrated system (Figure 3). Dengue virus and EV71 were mixed in the virus sample chamber, and then pumped into the pretreatment chamber. Lanes 1 and 2 are the results from the virus captured by anti-Dengue antibodyconjugated magnetic beads and amplified by the Dengue-specific and EV71-specific primers, respectively. Lanes 3 and 4 are the results from the virus captured by the anti-EV71 antibody-conjugated magnetic beads and amplified by the dengue-specific and EV71- specific primers, respectively. The results show that anti-Dengue antibody-conjugated magnetic beads only specifically captured the Dengue virus and amplified the Denguespecific detection gene, while the anti-EV71 antibody-conjugated beads only captured and amplified the EV71. The results demonstrate the specificity of the antibodyconjugated magnetic beads. Comparable to a large-scale apparatus, the new microsystem integrates the microfluidic module, the purification module, and the micro temperature control module, and can pretreat the biosamples and perform genetic diagnosis automatically in a short period of time and can become a crucial platform in biological and medical applications for rapid clinical diagnosis of viruses.
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