Browse > Article

Rapid Molecular Diagnosis using Real-time Nucleic Acid Sequence Based Amplification (NASBA) for Detection of Influenza A Virus Subtypes  

Lim, Jae-Won (Department of Biomedical Laboratory Science, College of Health Sciences, Yonsei University)
Lee, In-Soo (Department of Clinical Laboratory Science, Hyejeon Colleage)
Cho, Yoon-Jung (Department of Biomedical Laboratory Science, College of Health Sciences, Yonsei University)
Jin, Hyun-Woo (Department of Biomedical Laboratory Science, College of Health Sciences, Yonsei University)
Choi, Yeon-Im (Department of Biomedical Laboratory Science, College of Health Sciences, Yonsei University)
Lee, Hye-Young (Department of Biomedical Laboratory Science, College of Health Sciences, Yonsei University)
Kim, Tae-Ue (Department of Biomedical Laboratory Science, College of Health Sciences, Yonsei University)
Publication Information
Biomedical Science Letters / v.17, no.4, 2011 , pp. 297-304 More about this Journal
Abstract
Influenza A virus of the Orthomyxoviridae family is a contagious respiratory pathogen that continues to evolve and burden in the human public health. It is able to spread efficiently from human to human and have the potential to cause pandemics with significant morbidity and mortality. It has been estimated that every year about 500 million people are infected with this virus, causing about approximately 0.25 to 0.5 million people deaths worldwide. Influenza A viruses are classified into different subtypes by antigenicity based on their hemagglutinin (HA) and neuraminidase (NA) proteins. The sudden emergence of influenza A virus subtypes and access for epidemiological analysis of this subtypes demanded a rapid development of specific diagnostic tools. Also, rapid identification of the subtypes can help to determine the antiviral treatment, because the different subtypes have a different antiviral drug resistance patterns. In this study, our aim is to detect influenza A virus subtypes by using real-time nucleic acid sequence based amplification (NASBA) which has high sensitivity and specificity through molecular beacon. Real-time NASBA is a method that able to shorten the time compare to other molecular diagnostic tools and is performed by isothermal condition. We selected major pandemic influenza A virus subtypes, H3N2 and H5N1. Three influenza A virus gene fragments such as HA, NA and matrix protein (M) gene were targeted. M gene is distinguished influenza A virus from other influenza virus. We designed specific primers and molecular beacons for HA, NA and M gene, respectively. In brief, the results showed that the specificity of the real-time NASBA was higher than reverse transcription polymerase chain reaction (RT-PCR). In addition, time to positivity (TTP) of this method was shorter than real-time PCR. This study suggests that the rapid detection of neo-appearance pandemic influenza A virus using real-time NASBA has the potential to determine the subtypes.
Keywords
Influenza A virus; Real-time NASBA; RT-PCR;
Citations & Related Records
Times Cited By KSCI : 2  (Citation Analysis)
연도 인용수 순위
1 Loens K, Beck T, Ursi D, Overdijk M, Sillekens P, Goossens H, Ieven M. Development of real-time multiplex nucleic acid sequence-based amplification for detection of Mycoplasma pneumoniae, Chlamydophilia pneumoniae, and Legionella spp. in respiratory specimens. J Clin Microbiol. 2008. 46:185-191.   DOI   ScienceOn
2 Molden T, Kraus I, Skomedal H, Nordstrom T, Karlsen F. $PreTect^{TM}$ HPV-Proofer: Real-time detection and typing of E6/E7 mRNA from carcinogenic human papillomaviruses. J Virol Methods. 2007. 142: 204-212.   DOI   ScienceOn
3 Moore C, Telles JN, Corden S, Gao RB, Vernet G, Aarle PV, Shu YL. Development and validation a commercial real-time NASBA assay for the rapid confirmation of influenza A H5N1 virus in clinical samples. J Virol Methods. 2010. 170:173-176.   DOI   ScienceOn
4 Pabbaraju K, Wong S, Wong AA, Appleyard GD, Chui L, Pang XL, Yanow SK, Fonseca K, Lee BE, Fox JD, Preiksaitis JK. Design and validation of real-time reverse transcription-PCR assays for detection of pandemic (H1N1) 2009 virus. J Clin Microbiol. 2009. 47: 3454-3460.   DOI   ScienceOn
5 Park YH, Woo YD, Kim SK, Bae HJ, Park SW. Simultaneous detection and identification of human respiratory syncytial virus, influenza virus A (H3N2, H1N1) and B by one-tube multiplex reverse transcription polymerase chain reaction. J Bacteriol Virol. 2001. 31: 269-274.
6 Saxena SK, Mishra N, Saxena R, Swamy MLA, Sahgal P, Saxena S, Tiwari S, Mathur A, Nair MP. Structural and antigenic variance between novel influenza A/H1N1/2009 and influenza A/H1N1/2008 viruses. J Infect Dev Ctries. 2010. 4: 1-6.
7 Schulze M, Nitsche A, Schweiger B, Biere B. Diagnostic approach for the differentiation of the pandemic influenza A(H1N1)v virus from recent human influenza viruses by real-time PCR. PLoS ONE. 2010. 5: e9966.   DOI
8 Wiely DC, Skehel TT. Functional balance between hemagglutinin and neuraminidase in influenza virus infections. Ann Rev Biochem. 1987. 56: 365-394.   DOI   ScienceOn
9 Kim YY, Lee JY, Hwang JH, Kim KA, Jang SW, Park MS, Kim WJ, Cho HW, Lee HH, Kang C. Characterization of hemagglutinin and neuraminidase genes and Oseltamivir resistance of influenza viruses isolated Korea. J Bacteriol Virol. 2005. 35: 149-155.
10 Zhao J, Tang S, Storhoff J, Marla S, Bao YP, Wang X, Wong EY, Ragupathy W, Ye Z, Hewlett IK. Multiplexed, rapid detection of H5N1 using PCR-free nanoparticle-based genomic microarray assay. BMC Biotechnology. 2010. 10: 74-82.   DOI
11 Hwang YO, Seo BT, Choi BH. Analysis of isolation and subtyping of influenza virus in Seoul, during 1999-2003. J Bacteriol Virol. 2004. 34: 67-74.
12 Arias CF, Escalera-Zamudio M, Rio MD, Cobian-Guemes AG, Isa P, Lopez S. Molecular anatomy of 2009 influenza virus A (H1N1). J Arcmed. 2009. 40: 643-654.
13 Chidlow GR, Harnett GB, Williams SH, Tempone SS, Speers DJ, Hurt AC, Deng YM, Smith DW. The detection of Oseltamivir-resistant pandemic influenza A/H1N1 2009 viruses using a real-time RT-PCR assay. J Virol Methods. 2010. 169: 47-51.   DOI   ScienceOn
14 Ge Y, Cui L, Qi X, Shan J, Shan Y, Qi Y, Wu B, Wang H, Shi Z. Detection of novel swine origin influenza A virus (H1N1) by real-time nucleic acid sequence-based amplification. J Virol Methods. 2010. 163: 495-497.   DOI   ScienceOn
15 Kamps BS, Hoffmann C, Preiser W. Influenza report 2006. 2006. pp.18-22, 26-28, 87-91, 127-132, 150-155. Flying Publisher. Paris, France
16 Kim JS, Park KY, Lee MG, Kim YS, Ahn JB, Yoon HJ. Rapid and type-specific detection of human influenza viruses using reverse transcription-polymerase chain reaction. J Korean Soc Microbiol. 1995. 30: 233-243.