A multiplex PCR assay for the detection of five influenza viruses using a dual priming oligonucleotide system

BMC Infectious Diseases

Kongxin Hu¹  Wei Zhen¹  Xiaohong Sun¹  Liping Zhang¹  Pengfei Yang³  Lei Shi²  Huanzhou Xu¹  Xuezheng Ma¹ 

[1]Institute of Health and Quarantine, Chinese Academy of Inspection and Quarantine, |No.A3, Gaobeidian North Road, Beijing 100123, Chaoyang District, China

[2]Department of Disease Control and Prevention, Shenzhen International Travel Health Care Center, Shenzhen 518045, Guangdong Province, China

[3]Huaian Center for Disease Control and Prevention, No.118, Huaihai North Road, Qinghe District, Huaian, Jiangsu Province, China

关键词: Influenza;    Multiplex PCR;    DPO;    Dual priming oligonucleotide;   

Others  :  1135699
DOI  :  10.1186/s12879-015-0818-y

 received in 2014-08-04, accepted in 2015-02-10,  发布年份 2015

【 摘 要 】

Background

A cost-effective, accurate and rapid simultaneous multiplex assay is required for testing and diagnoses of conventional and emerging viruses in clinical virology laboratories. We developed and optimized a dual priming oligonucleotide (DPO) multiplex PCR assay for detecting influenza viruses including seasonal H1N1, 2009 pandemic H1N1, H3N2, influenza B and H5N1.

Methods

The optimized multiplex DPO PCR was used to detect 233 clinical human samples. The results were compared to those obtained with RT-qPCR, conventional PCR and immunochromatographic assay.

Results

Specificity analysis revealed that the DPO PCR assay amplified each target virus without any cross-amplification. Statistical analysis demonstrated that the multiplex DPO-PCR sensitivity was higher than for the immunochromatographic assay and lower than for qPCR, while no significant difference was observed compared with conventional PCR, when detecting influenza A and B. Additional experiments using the same sample panel indicated no significant differences between the number of positive samples detected by multiplex DPO PCR and RT-qPCR when applying a Cq with a value lower than 30.

Conclusions

The five-targeted simultaneous multiplex DPO PCR assay could be easily adopted into routine practice. This approach is cost effective with a short running time, low technical requirements for the detection of influenza virus and early diagnosis in clinical laboratories.

【 授权许可】

   
2015 Ma et al.; licensee BioMed Central.

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【 参考文献 】

• [1]Simonsen L, Spreeuwenberg P, Lustig R, Taylor RJ, Fleming DM, Kroneman M, et al.: GLaMOR Collaborating Teams. Global mortality estimates for the 2009 Influenza Pandemic from the GLaMOR project: a modeling study. PLoS Med 2009, 2013(10):1-17.
• [2]Arya M, Shergill IS, Williamson M, Gommersall L, Arya N, Patel HR: Basic principles of real-time quantitative PCR. Expert Rev Mol Diagn. 2005, 5:209-19.
• [3]Kim HK, Oh SH, Yun KA, Sung H, Kim MN: Comparison of Anyplex II RV16 with the xTAG respiratory viral panel and Seeplex RV15 fordetection of respiratory viruses. J Clin Microbiol. 2013, 51:1137-41.
• [4]Bibby DF, McElarney I, Breuer J, Clark DA: Comparative evaluation of the Seegene Seeplex RV15 and real-time PCR for respiratory virus detection. J Med Virol. 2011, 83:1469-75.
• [5]Kim SR, Ki CS, Lee NY: Rapid detection and identification of 12 respiratory viruses using a dual priming oligonucleotide system-based multiplex PCR assay. J Virol Methods. 2009, 156:111-6.
• [6]Lee CS, Kang BK, Lee DH, Lyou SH, Park BK, Ann SK, et al.: One-step multiplex RT-PCR for detection and subtyping of swine influenza H1, H3, N1, N2 viruses in clinical samples using a dual priming oligonucleotide (DPO) system. J Virol Methods. 2008, 151:30-4.
• [7]Yoo SJ, Kuak EY, Shin BM: Detection of 12 respiratory viruses with two-set multiplex reverse transcriptase-PCR assay using a dual priming oligonucleotide system. Korean J Lab Med. 2007, 27:420-7.
• [8]Li C, Bu Z, Chen H: Avian influenza vaccines against H5N1 'bird flu'. Trends Biotechnol. 2014, 432:147-56.
• [9]Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S: MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol. 2011, 28:2731-9.
• [10]WHO: WHO information for molecular diagnosis of influenza virus in humans - update. 2012.
• [11]Chun JY, Kim KJ, Hwang IT, Kim YJ, Lee DH, Lee IK, et al.: Dual priming oligonucleotide system for the multiplex detection of respiratory viruses and SNP genotyping of CYP2C19 gene. Nucleic Acids Res. 2007, 35:1-6.
• [12]Kim H, Hur M, Moon HW, Yun YM, Cho HC: Comparison of two multiplex PCR assays for the detection of respiratory viral infections. Clin Respir J. 2014, 8:391-6.
• [13]Cho CH, Chulten B, Lee CK, Nam MH, Yoon SY, Lim CS, et al.: Evaluation of a novel real-time RT-PCR using TOCE technology compared with culture and Seeplex RV15 forsimultaneous detection of respiratory viruses. J Clin Virol. 2013, 57:338-42.
• [14]Li L, Chen QY, Li YY, Wang YF, Yang ZF, Zhong NS: Comparison among nasopharyngeal swab, nasal wash, and oropharyngeal swab for respiratory virus detection in adults with acute pharyngitis. BMC Infect Dis. 2013, 13:281. BioMed Central Full Text
• [15]Centers for Disease Control and Prevention: Evaluation of 11 commercially available rapid influenza diagnostic tests--United States, 2011–2012 MMWR Morb Mortal Wkly Rep 2012, 61:873-6.
• [16]Loens K, Van Heirstraeten L, Malhotra-Kumar S, Goossens H, Ieven M: Optimal sampling sites and methods for detection of pathogens possibly causing community-acquired lower respiratory tract infections. J Clin Microbiol. 2009, 47:21-31.
• [17]Hindiyeh M, Levy V, Azar R, Varsano N, Regev L, Shalev Y, et al.: Evaluation of a multiplex real-time reverse transcriptase PCR assay for detection and differentiation of influenza viruses A and B during the 2001–2002 influenza season in Israel. J Clin Microbiol. 2005, 43:589-95.