Detection of human novel influenza A (H1N1) viruses using multi-fluorescent real-time RT-PCR.
The novel influenza A (H1N1) virus is now rapidly spreading across the world. Early detection is one of the most effective measures to prevent further transmission of the virus. 4 sets of proprietary primers and probes designed for detection of influenza A viruses (InfA), human and swine H1N1 viruses (SH1), the novel H1N1 viruses (NH1) and RNaseP gene (RP) respectively were pooled together in a single tube for a multi-fluorescent real-time RT-PCR assay. The detection limit was found to be one order more sensitive than that employing the WHO recommended protocol. The NH1 probe was negative for all control samples including human seasonal H1N1 virus, other subtypes of human influenza A viruses (H3, H5, H9), human influenza B virus and nasopharyngeal swabs from patients with noninfluenza respiratory diseases, indicating its high specificity, capable of discriminating the novel influenza A virus from the previously identified H1N1 viruses. For confirmation, the PCR amplified fragment of the hemagglutinin gene was sequenced which could provide enough information to identify the novel H1N1 virus as a distinct cluster among all viruses of subtype H1 through average distance clustering analysis. Although these assays should be useful in the current outbreak for rapid detection and discrimination of the novel H1N1 from swine H1N1 and other human seasonal H1N1 viruses, further design improvement is suggested to match possible sequence variations in the detected region along with the course of the epidemic.
Dong
Hui
H
Shanghai-MOST Key Laboratory for Disease and Health Genomics, Chinese National Human Genome Center at Shanghai, Shanghai 201203, China.
Zhang
Yanjun
Y
Xiong
Hui
H
Yan
An
A
Ding
Guohui
G
Chen
Yangyi
Y
Xie
Liqun
L
Chen
Jiazheng
J
Zhang
Guoqing
G
Hao
Pei
P
Cong
Liming
L
Lu
Yiyu
Y
Che
Xiaoyan
X
Wang
Xiaoning
X
Li
Yixue
Y
Yuen
Kwok-Yung
KY
Zhao
Guoping
G
Jin
Weirong
W
D016000:Cluster Analysis; D017931:DNA Primers / Q000235:genetics; D005838:Genotype; D006389:Hemagglutinins, Viral / Q000235:genetics; D006801:Humans; D053118:Influenza A Virus, H1N1 Subtype / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D007251:Influenza, Human / Q000175:diagnosis* / Q000821:virology*; D012367:RNA, Viral / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D017385:Sequence Homology
D017931:DNA Primers; D006389:Hemagglutinins, Viral; D012367:RNA, Viral; C458881:hemagglutinin fusogenic peptide, influenza virus