Publish in OALib Journal
ISSN: 2333-9721
APC: Only $99

Paper Submission

Views	Downloads

Relative Articles
Clinical and Virological Factors Associated with Viremia in Pandemic Influenza A/H1N1/2009 Virus Infection
Prediction of Biological Functions on Glycosylation Site Migrations in Human Influenza H1N1 Viruses
Diagnostic Accuracy of a Rapid Influenza Test for Pandemic Influenza A H1N1
Age Distribution of Cases of 2009 (H1N1) Pandemic Influenza in Comparison with Seasonal Influenza
The investigation of Risk factors of influenza pandemic H1N1
2009 H1N1 Influenza 2009 H1N1 Influenza
Pandemic H1N1 influenza infections in 2009
Improving the Clinical Diagnosis of Influenza—a Comparative Analysis of New Influenza A (H1N1) Cases
An Epidemiological Study on Influenza A(H1N1) in Makkah
Pandemic influenza A/H1N1: comparative analysis of microscopic lung histopathological findings
More...

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

Hyun-Ok Song, Je-Hyoung Kim, Ho-Sun Ryu, Dong-Hoon Lee, Sun-Jin Kim, Deog-Joong Kim, In Bum Suh, Du Young Choi, Kwang-Ho In, Sung-Woo Kim, Hyun Park

Full-Text Cite this paper Add to My Lib

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.

References

[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.

Full-Text