[Colorimetric detection of human influenza A H1N1 virus by reverse transcription loop mediated isothermal amplification].
A simple, rapid and sensitive colorimetric Reverse Transcription Loop Mediated Isothermal Amplification (RT-LAMP) method was established to detect human influenza A H1N1 virus. The method employed a set of six specially designed primers that recognized eight distinct sequences of the HA gene for amplification of nucleic acid under isothermal conditions at 65 degrees C for one and half hour. The amplification process of RT-LAMP was monitored by the addition of HNB (Hydroxy naphthol blue) dye prior to amplification. A positive reaction was indicated by a color change from violet to sky blue and confirmed by agarose electrophoresis. The specificity of the RT-LAMP assay was validated by cross-reaction with different swine and human influenza virus including human seasonal influenza A /H1N1 A /H3N2, influenza B and swine A /H1N1. The sensitivity of this assay was evaluated by serial dilutions of RNA molecules from in vitro transcription of human influenza A H1N1 HA gene. The assay was further evaluated with 30 clinical specimens with suspected pandemic influenza A H1N1 virus infection in parallel with RT-PCR detection and 26 clinical specimens with seasonal influenza virus infection. Our results showed that the RT-LAMP was able to achieve a sensitivity of 60 RNA copies with high specificity, and detection rate was comparable to that of the RT-PCR with the clinical samples of pandemic influenza A H1N1 infection. The RT-LAMP reaction with HNB could also be measured at 650nm in a microplate reader for quantitative analysis. Thus, we concluded that this colorimetric RT-LAMP assay had potential for the rapid screening of the human influenza A H1N1 virus infection in National influenza monitoring network laboratories and sentinel hospitals of provincial and municipal region in China.
Nie
Kai
K
State Key Laboratory for molecular Virology and Genetic Engineering, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 100052, China.
Wang
Da-Yan
DY
Qin
Meng
M
Gao
Rong-Bao
RB
Wang
Miao
M
Zou
Shu-Mei
SM
Han
Feng
F
Zhao
Xiang
X
Li
Xi-Yan
XY
Shu
Yue-Long
YL
Ma
Xue-Jun
XJ
D000818:Animals; D003124:Colorimetry / Q000379:methods; D017931:DNA Primers / Q000235:genetics; D004587:Electrophoresis, Agar Gel; 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; D009282:Naphthalenesulfonates / Q000737:chemistry; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D009976:Orthomyxoviridae Infections / Q000175:diagnosis / Q000662:veterinary / Q000821:virology; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013552:Swine; D013553:Swine Diseases / Q000175:diagnosis / Q000821:virology; D013696:Temperature
D017931:DNA Primers; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D009282:Naphthalenesulfonates; C540250:trisodium 3-hydroxy-4-((2Z)-2-(2-oxo-4-sulfonatonaphthalen-1-ylidene)hydrazinyl)naphthalene-2,7-disulfonate