Molecular detection of a novel human influenza (H1N1) of pandemic potential by conventional and real-time quantitative RT-PCR assays.
BACKGROUND
Influenza A viruses are medically important viral pathogens that cause significant mortality and morbidity throughout the world. The recent emergence of a novel human influenza A virus (H1N1) poses a serious health threat. Molecular tests for rapid detection of this virus are urgently needed.


METHODS
We developed a conventional 1-step RT-PCR assay and a 1-step quantitative real-time RT-PCR assay to detect the novel H1N1 virus, but not the seasonal H1N1 viruses. We also developed an additional real-time RT-PCR that can discriminate the novel H1N1 from other swine and human H1 subtype viruses.


RESULTS
All of the assays had detection limits for the positive control in the range of 1.0 x 10(-4) to 2.0 x 10(-3) of the median tissue culture infective dose. Assay specificities were high, and for the conventional and real-time assays, all negative control samples were negative, including 7 human seasonal H1N1 viruses, 1 human H2N2 virus, 2 human seasonal H3N2 viruses, 1 human H5N1 virus, 7 avian influenza viruses (HA subtypes 4, 5, 7, 8, 9, and 10), and 48 nasopharyngeal aspirates (NPAs) from patients with noninfluenza respiratory diseases; for the assay that discriminates the novel H1N1 from other swine and human H1 subtype viruses, all negative controls were also negative, including 20 control NPAs, 2 seasonal human H1N1 viruses, 2 seasonal human H3N2 viruses, and 2 human H5N1 viruses.


CONCLUSIONS
These assays appear useful for the rapid diagnosis of cases with the novel H1N1 virus, thereby allowing better pandemic preparedness.
Department of Microbiology, The University of Hong Kong, Hong Kong SAR, China.
Leo L M
LL
Poon
K H
KH
Chan
G J
GJ
Smith
C S W
CS
Leung
Y
Y
Guan
K Y
KY
Yuen
J S M
JS
Peiris
D000818:Animals; D001483:Base Sequence; D004279:DNA, Viral / Q000032:analysis / Q000235:genetics; D006801:Humans; D053118:Influenza A Virus, H1N1 Subtype / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D053121:Influenza A Virus, H2N2 Subtype / Q000145:classification / Q000235:genetics / Q000302:isolation & purification; D053122:Influenza A Virus, H3N2 Subtype / Q000145:classification / Q000235:genetics / Q000302:isolation & purification; D053124:Influenza A Virus, H5N1 Subtype / Q000145:classification / Q000235:genetics / Q000302:isolation & purification; D007251:Influenza, Human / Q000175:diagnosis* / Q000821:virology; D025202:Molecular Diagnostic Techniques / Q000191:economics / Q000379:methods*; D009976:Orthomyxoviridae Infections / Q000175:diagnosis* / Q000821:virology; D012367:RNA, Viral / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000191:economics / Q000379:methods*; D012680:Sensitivity and Specificity; D013552:Swine / Q000821:virology*; D013997:Time Factors
D004279:DNA, Viral; D012367:RNA, Viral