Abstract
Recently, human deaths have resulted from infection with low-pathogenicity avian influenza virus H7N9 strains that have emerged recently in China. To strengthen H7N9 surveillance and outbreak control, rapid and reliable diagnostic methods are needed. To develop a sensitive quantitative real-time RT-PCR assay for rapid detection of H7N9 viral RNA, primers and AllGlo probes were designed to target the HA and NA genes of H7N9. Conserved sequences in the HA and NA genes were identified by phylogenic analysis and used as targets for H7N9 virus detection. The similarities of the targeted HA and NA gene sequences from different H7 and N9 influenza virus strains were 93.2-99.9 % and 96.0-99.6 %, respectively The specificity and sensitivity of the new multiplex real-time qRT-PCR was established. The test was used for the detection of viral RNA in human pharyngeal swabs and environmental samples. The detection limit of the multiplex qRT-PCR was estimated to be about 10−1 TCID50/reaction. Finally, the diagnostic sensitivities of the multiplex qRT-PCR, virus isolation and TaqMan qRT-PCR were compared using pharyngeal swabs and environmental samples. These analyses yielded positive results in 46.7 %, 43.3 % and 20.0 % of the samples, respectively. The novel multiplex AllGlo qRT-PCR is a rapid and sensitive method to identify H7N9 virus in clinical and environmental samples and can be used to facilitate studies on the epidemiology of H7N9 virus.
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References
Gao R, Cao B, Hu Y, Feng Z, Wang D, Hu W, Chen J, Jie Z, Qiu H, Xu K, Xu X, Lu H, Zhu W, Gao Z, Xiang N, Shen Y, He Z, Gu Y, Zhang Z, Yang Y, Zhao X, Zhou L, Li X, Zou S, Zhang Y, Li X, Yang L, Guo J, Dong J, Li Q, Dong L, Zhu Y, Bai T, Wang S, Hao P, Yang W, Zhang Y, Han J, Yu H, Li D, Gao GF, Wu G, Wang Y, Yuan Z, Shu Y (2013) Human infection with a novel avian-origin influenza A (H7N9) virus. N Engl J Med 368:1888–1897
Chen Y, Liang W, Yang S, Wu N, Gao H, Sheng J, Yao H, Wo J, Fang Q, Cui D, Li Y, Yao X, Zhang Y, Wu H, Zheng S, Diao H, Xia S, Zhang Y, Chan KH, Tsoi HW, Teng JL, Song W, Wang P, Lau SY, Zheng M, Chan JF, To KK, Chen H, Li L, Yuen KY (2013) Human infections with the emerging avian influenza A H7N9 virus from wet market poultry: clinical analysis and characterisation of viral genome. Lancet 381:1916–1925
Gao HN, Lu HZ, Cao B, Du B, Shang H, Gan JH, Lu SH, Yang YD, Fang Q, Shen YZ, Xi XM, Gu Q, Zhou XM, Qu HP, Yan Z, Li FM, Zhao W, Gao ZC, Wang GF, Ruan LX, Wang WH, Ye J, Cao HF, Li XW, Zhang WH, Fang XC, He J, Liang WF, Xie J, Zeng M, Wu XZ, Li J, Xia Q, Jin ZC, Chen Q, Tang C, Zhang ZY, Hou BM, Feng ZX, Sheng JF, Zhong NS, Li LJ (2013) Clinical findings in 111 cases of influenza A (H7N9) virus infection. N Engl J Med 368:2277–2285
Zhang L, Ding G, Wei L, Pan X, Mei L, Zhang Y, Lu Y (2011) Establishment of a novel target-based real-time quantitative PCR method for Acinetobacter baumannii detection. Diagn Mol Pathol 20:242–248
Ge Y, Wu B, Qi X, Zhao K, Guo X, Zhu Y, Qi Y, Shi Z, Zhou M, Wang H, Cui L (2013) Rapid and sensitive detection of novel avian-origin influenza A (H7N9) virus by reverse transcription loop-mediated isothermal amplification combined with a lateral-flow device. PLoS One 8:e69941
Corman VM, Eickmann M, Landt O, Bleicker T, Brünink S, Eschbach-Bludau M, Matrosovich M, Becker S, Drosten C (2013) Specific detection by real-time reverse-transcription PCR assays of a novel avian influenza A(H7N9) strain associated with human spillover infections in China. Euro Surveill 18:20461
Yu D, Chen Y, Wu S, Wang B, Tang YW, Li L (2012) Simultaneous detection and differentiation of human papillomavirus genotypes 6, 11, 16 and 18 by AllGloquadruplex quantitative PCR. PLoS One 7:e48972
Feng ZL, Yu XY, Lu ZM, Geng DY, Zhang L, Chen SJ (2011) Rapid detection of the hepatitis B virus YMDD mutant using AllGlo™ probes. Clin Chim Acta 412:1018–1021
Kim HR, Park CK, Oem JK, Bae YC, Choi JG, Lee OS, Lee YJ (2010) Characterization of H5N2 influenza viruses isolated in South Korea and their influence on the emergence of a novel H9N2 influenza virus. J Gen Virol 91:1978–1983
Choudhary ML, Anand SP, Heydari M, Rane G, Potdar VA, Chadha MS, Mishra AC (2013) Development of a multiplex one step RT-PCR that detects eighteen respiratory viruses in clinical specimens and comparison with real time RT-PCR. J Virol Methods 189:15–19
Acknowledgments
This work was supported by grants from the Provincial Medical Research Fund of Zhejiang, China (WKJ2011-2-016), the Zhejiang Provincial Program for the Cultivation of High-Level Innovative Health Talents, and Monitor Technology Platform of Infectious Diseases of the State Major Science and Technology Special Projects during the 12th Five-Year Plan of China (2012ZX10004-210).
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The authors declare that they have no competing interests.
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Y. Zhang, H. Mao, J. Yan, X. Wang and L. Zhang contributed equally to this article.
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Zhang, Y., Mao, H., Yan, J. et al. Development of novel AllGlo-probe-based one-step multiplex qRT-PCR assay for rapid identification of avian influenza virus H7N9. Arch Virol 159, 1707–1713 (2014). https://doi.org/10.1007/s00705-014-1979-5
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DOI: https://doi.org/10.1007/s00705-014-1979-5