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PLOS ONE  2012 

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

DOI: 10.1371/journal.pone.0053325

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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.


[1]  Dawood FS, Jain S, Finelli L, Shaw MW, Lindstrom S, et al. (2009) Emergence of a novel swine-origin influenza A (H1N1) virus in humans. N Engl J Med 360: 2605–2615.
[2]  Maines TR, Jayaraman A, Belser JA, Wadford DA, Pappas C, et al. (2009) Transmission and pathogenesis of swine-origin 2009 A(H1N1) influenza viruses in ferrets and mice. Science 325: 484–487.
[3]  Munster VJ, de Wit E, van den Brand JM, Herfst S, Schrauwen EJ, et al. (2009) Pathogenesis and transmission of swine-origin 2009 A(H1N1) influenza virus in ferrets. Science 325: 481–483.
[4]  Horimoto T, Kawaoka Y (2001) Pandemic threat posed by avian influenza A viruses. Clin Microbiol Rev 14: 129–149.
[5]  Fouchier RA, Bestebroer TM, Herfst S, Van Der Kemp L, Rimmelzwaan GF, et al. (2000) Detection of influenza A viruses from different species by PCR amplification of conserved sequences in the matrix gene. J Clin Microbiol 38: 4096–4101.
[6]  Landry ML (2011) Diagnostic tests for influenza infection. Curr Opin Pediatr 23: 91–97.
[7]  Evaluation of rapid influenza diagnostic tests for detection of novel influenza A (H1N1) Virus - United States, 2009. MMWR Morb Mortal Wkly Rep 58: 826–829.
[8]  Uyeki T (2009) Diagnostic testing for 2009 pandemic influenza A (H1N1) virus infection in hospitalized patients. N Engl J Med 361: e114.
[9]  Kok J, Blyth CC, Foo H, Patterson J, Taylor J, et al. (2010) Comparison of a rapid antigen test with nucleic acid testing during cocirculation of pandemic influenza A/H1N1 2009 and seasonal influenza A/H3N2. J Clin Microbiol 48: 290–291.
[10]  Faix DJ, Sherman SS, Waterman SH (2009) Rapid-test sensitivity for novel swine-origin influenza A (H1N1) virus in humans. N Engl J Med 361: 728–729.
[11]  Hurt AC, Baas C, Deng YM, Roberts S, Kelso A, et al. (2009) Performance of influenza rapid point-of-care tests in the detection of swine lineage A(H1N1) influenza viruses. Influenza Other Respi Viruses 3: 171–176.
[12]  Performance of rapid influenza diagnostic tests during two school outbreaks of 2009 pandemic influenza A (H1N1) virus infection - Connecticut, 2009. MMWR Morb Mortal Wkly Rep 58: 1029–1032.
[13]  Chan KH, Lai ST, Poon LL, Guan Y, Yuen KY, et al. (2009) Analytical sensitivity of rapid influenza antigen detection tests for swine-origin influenza virus (H1N1). J Clin Virol 45: 205–207.
[14]  Hawkes M, Richardson SE, Ipp M, Schuh S, Adachi D, et al. (2010) Sensitivity of rapid influenza diagnostic testing for swine-origin 2009 a (H1N1) influenza virus in children. Pediatrics 125: e639–644.
[15]  Andresen DN, Kesson AM (2010) High sensitivity of a rapid immunochromatographic test for detection of influenza A virus 2009 H1N1 in nasopharyngeal aspirates from young children. J Clin Microbiol 48: 2658–2659.
[16]  Mackay IM, Arden KE, Nitsche A (2002) Real-time PCR in virology. Nucleic Acids Res 30: 1292–1305.
[17]  Carr MJ, Gunson R, Maclean A, Coughlan S, Fitzgerald M, et al. (2009) Development of a real-time RT-PCR for the detection of swine-lineage influenza A (H1N1) virus infections. J Clin Virol 45: 196–199.
[18]  Jiang T, Kang X, Deng Y, Zhao H, Li X, et al. (2010) Development of a real-time RT-PCR assay for a novel influenza A (H1N1) virus. J Virol Methods 163: 470–473.
[19]  Pabbaraju K, Wong S, Wong AA, Appleyard GD, Chui L, et al. (2009) Design and validation of real-time reverse transcription-PCR assays for detection of pandemic (H1N1) 2009 virus. J Clin Microbiol 47: 3454–3460.
[20]  Wang R, Sheng ZM, Taubenberger JK (2009) Detection of novel (swine origin) H1N1 influenza A virus by quantitative real-time reverse transcription-PCR. J Clin Microbiol 47: 2675–2677.
[21]  Whiley DM, Bialasiewicz S, Bletchly C, Faux CE, Harrower B, et al. (2009) Detection of novel influenza A(H1N1) virus by real-time RT-PCR. J Clin Virol 45: 203–204.
[22]  Yang Y, Gonzalez R, Huang F, Wang W, Li Y, et al. (2010) Simultaneous typing and HA/NA subtyping of influenza A and B viruses including the pandemic influenza A/H1N1 2009 by multiplex real-time RT-PCR. J Virol Methods 167: 37–44.
[23]  Hall RJ, Peacey M, Huang QS, Carter PE (2009) Rapid method to support diagnosis of swine origin influenza virus infection by sequencing of real-time PCR amplicons from diagnostic assays. J Clin Microbiol 47: 3053–3054.
[24]  Kawai Y, Kimura Y, Lezhava A, Kanamori H, Usui K, et al. (2012) One-step detection of the 2009 pandemic influenza A(H1N1) virus by the RT-SmartAmp assay and its clinical validation. PLoS One 7: e30236.
[25]  Munchow G, Dadic D, Doffing F, Hardt S, Drese KS (2005) Automated chip-based device for simple and fast nucleic acid amplification. Expert Rev Mol Diagn 5: 613–620.
[26]  Zhang C, Xu J, Ma W, Zheng W (2006) PCR microfluidic devices for DNA amplification. Biotechnol Adv 24: 243–284.
[27]  Frey O, Bonneick S, Hierlemann A, Lichtenberg J (2007) Autonomous microfluidic multi-channel chip for real-time PCR with integrated liquid handling. Biomed Microdevices 9: 711–718.
[28]  Zhang C, Xing D (2007) Miniaturized PCR chips for nucleic acid amplification and analysis: latest advances and future trends. Nucleic Acids Res 35: 4223–4237.
[29]  Chen Z, Mauk MG, Wang J, Abrams WR, Corstjens PL, et al. (2007) A microfluidic system for saliva-based detection of infectious diseases. Ann N Y Acad Sci 1098: 429–436.
[30]  Hagan KA, Reedy CR, Uchimoto ML, Basu D, Engel DA, et al. (2011) An integrated, valveless system for microfluidic purification and reverse transcription-PCR amplification of RNA for detection of infectious agents. Lab Chip 11: 957–961.
[31]  Sun Y, Dhumpa R, Bang DD, Hogberg J, Handberg K, et al. (2011) A lab-on-a-chip device for rapid identification of avian influenza viral RNA by solid-phase PCR. Lab Chip 11: 1457–1463.
[32]  Teo J, Di Pietro P, San Biagio F, Capozzoli M, Deng YM, et al. (2011) VereFlu: an integrated multiplex RT-PCR and microarray assay for rapid detection and identification of human influenza A and B viruses using lab-on-chip technology. Arch Virol 156: 1371–1378.
[33]  Becker H, Locascio LE (2002) Polymer microfluidic devices. Talanta 56: 267–287.
[34]  Mair DA, Geiger E, Pisano AP, Frechet JM, Svec F (2006) Injection molded microfluidic chips featuring integrated interconnects. Lab Chip 6: 1346–1354.
[35]  Hoffmann E, Stech J, Guan Y, Webster RG, Perez DR (2001) Universal primer set for the full-length amplification of all influenza A viruses. Arch Virol 146: 2275–2289.


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