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

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 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 5 x 10 –2 50 % egg infective doses (EID 50 ). In contrast, the minimum detection limit was approximately 3 EID 50 in conventional RT-PCR with WHO primers and 10 EID 50 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 4 x 10 8 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: EID 50 , 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. These authors contributed equally to this paper.