[Development of single-tube multiplex real-time PCR for simultaneous detection of novel influenza A H1N1 and human seasonal influenza A H1N1 and H3N2 virus].
In this study, we established a rapid and sensitive multiplex Real-time reverse transcription polymerase chain reaction (mrtRT-PCR) to simultaneously detect the novel human influenza A H1N1 virus, human seasonal influenza A H1N1 and H3N2. This assay had three pairs of primer to target the conserved regions of the HA gene for each of the HA types including novel H1N1, seasonal H1N1 and seasonal H3N2, and one pair of primer designed to detect the internal control-RnaseP gene. This assay was performed in one-step in one tube. To validate the specificity of the multiplex Real-time RT-PCR assay, different human influenza virus including human seasonal influenza A H1N1 and H3N2, human influenza B and reference A/California/07/2009 (H1N1) sw1 was tested. To evaluate the sensitivity of the assay, serial dilutions of RNA from in vitro transcription of the novel human influenza A H1N1 HA gene was tested. Finally this assay was evaluated with clinical samples from 54 fever patients diagnosed with novel influenza A H1N1 or seasonal H1/H3 or HB infection either by real-time PCR recommended by the WHO or HI assay by the National Influenza Center. Our results showed that the assay could achieve a sensitivity of 20 RNA copies of novel influenza A H1N1 with high specificity and could detect potential mixed co-infection. In conclusion, this multiplex real-time RT-PCR assay combines both rapidity and sensitivity for not only detecting the novel human influenza A H1N1 virus, but also monitoring the human seasonal influenza A H1N1 and H3N2 simultaneously.
Qin
Meng
M
National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 100052, China. alex2003@yeah.net
Wang
Da-Yan
DY
Huang
Fang
F
Nie
Kai
K
Qu
Mei
M
Wang
Miao
M
Han
Feng
F
Zhao
Xiang
X
Cheng
Yan-Hui
YH
Shu
Yue-Long
YL
Ma
Xue-Jun
XJ
D017931:DNA Primers / Q000235:genetics; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000235:genetics; D006801:Humans; D053118:Influenza A Virus, H1N1 Subtype / Q000235:genetics*; D053122:Influenza A Virus, H3N2 Subtype / Q000235:genetics*; D007251:Influenza, Human / Q000175:diagnosis* / Q000821:virology; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods*; D012621:Seasons; D012680:Sensitivity and Specificity
D017931:DNA Primers; D019267:Hemagglutinin Glycoproteins, Influenza Virus