Real-time RT-PCR for H5N1 avian influenza A virus detection.
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.
Chen
Weijun
W
Beijing Genomics Institute, Chinese Academy of Sciences, Beijing, China.
He
Bo
B
Beijing Genomics Institute, Chinese Academy of Sciences, Beijing, China.
Li
Changgui
C
National Institute for the Control of Pharmaceutical and Biological Products, Beijing, China.
Zhang
Xiaowei
X
Beijing Genomics Institute, Chinese Academy of Sciences, Beijing, China.
Wu
Weili
W
Beijing Genomics Institute, Chinese Academy of Sciences, Beijing, China.
Yin
Xuyang
X
Wenzhou Medical College, Wenzhou, China.
Fan
Baoxing
B
Beijing 301 Hospital, Beijing, China.
Fan
Xingliang
X
National Institute for the Control of Pharmaceutical and Biological Products, Beijing, China.
Wang
Jian
J
Beijing Genomics Institute, Chinese Academy of Sciences, Beijing, China.
D000818:Animals; D001717:Birds; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D053124:Influenza A Virus, H5N1 Subtype / Q000235:genetics / Q000302:isolation & purification*; D005585:Influenza in Birds / Q000821:virology*; D007251:Influenza, Human / Q000821:virology*; D010614:Pharynx / Q000821:virology; D012367:RNA, Viral / Q000032:analysis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D019562:Viral Load
D012367:RNA, Viral