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J Med Microbiol 56 (2007), 603-607; DOI: 10.1099/jmm.0.47014-0
© 2007 Society for General Microbiology
ISSN 1473-5644

Real-time RT-PCR for H5N1 avian influenza A virus detection

Weijun Chen1,{dagger}, Bo He1,{dagger}, Changgui Li2,{dagger}, Xiaowei Zhang1, Weili Wu1, Xuyang Yin3, Baoxing Fan4, Xingliang Fan2 and Jian Wang1

1 Beijing Genomics Institute, Chinese Academy of Sciences, Beijing, China

2 National Institute for the Control of Pharmaceutical and Biological Products, Beijing, China

3 Wenzhou Medical College, Wenzhou, China

4 Beijing 301 Hospital, Beijing, China

Correspondence
Weijun Chen
chenwj{at}genomics.org.cn
Jian Wang
wangjian{at}genomics.org.cn

Received 20 October 2006
Accepted 11 January 2007

The recent recurrence of highly pathogenic avian influenza virus A H5N1 was firstly reported in mid-December 2003 and continued through 2005. This study describes a sensitive and specific real-time RT-PCR method for the detection of influenza A subtype H5 and for monitoring virus loads. Using serial dilutions of influenza A H5N1 cultures, this assay reproducibly determined the lowest detection limit to be approximately 5x10–2 50 % egg infective doses (EID50). In contrast, the minimum detection limit was approximately 3 EID50 in conventional RT-PCR with WHO primers and 10 EID50 in antigen-capture ELISA. In tests of serial dilutions of in vitro-transcribed influenza A H5 gene RNA, there was linear amplification from 40 copies to 4x108 copies of target RNA per reaction and approximately six copies, and sometimes even as few as three copies, of target RNA tested positive in our assay. Thirty-five throat swabs from ill birds were tested: 33 samples tested positive using this assay. In comparison, 27, 13 and 19 samples tested positive using conventional RT-PCR, antigen-capture ELISA and virus isolation, respectively. To evaluate further the sensitivity of this real-time RT-PCR, a standard panel and 60 H5N1 isolates that contained different clades of influenza virus A/H5N1 were tested and all tested positive. To evaluate the specificity of the assay, 60 throat swabs from patients infected with influenza virus A H1 were tested; all were negative. Thirteen other viruses were also tested and all tested negative.

Abbreviations: EID50, 50 % egg infective dose; FAM, 6-carboxyfluorescein; HA, haemagglutinin; HPAIV, highly pathogenic avian influenza virus; RRT-PCR, real-time RT-PCR; TAMRA, 6-carboxytetramethylrhodamine; WHO, World Health Organization.

{dagger}These authors contributed equally to this paper.




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