title: Evaluation of twelve real-time reverse transcriptase PCR primer-probe sets for detection of pandemic influenza A/H1N1 2009 virus.
abstract: Real-time reverse transcriptase PCR (rRT-PCR) assays have greatly contributed to the detection, control, and prevention of the pandemic influenza A/H1N1 2009 virus. To improve the rRT-PCR assays for detection of pandemic influenza A/H1N1 2009 virus, we evaluated the sensitivity, specificity, and performance of 12 rRT-PCR primer-probe sets [SW (a) to SW (l)] using a panel of virus strains and clinical specimens. These primer-probe sets were derived from published work and designed for detecting the hemagglutinin (HA) or the neuraminidase (NA) gene of the pandemic influenza A/H1N1 2009 virus. A primer-probe set, SW (CDC), developed by the Centers for Disease Control and Prevention (U.S. CDC) to target the HA gene of pandemic influenza A/H1N1 2009 virus, was used as a referee method. Our results demonstrated that although all primer-probe sets in this study had as high as 98.4 to 100% in silico coverage, some of the primer-probe sets had better specificity, sensitivity, and amplification efficiency than others. Two primer-probe sets, SW (h) and SW (l), which target the NA gene of pandemic influenza A/H1N1 2009 virus, were highly sensitive (10(4) copies/reaction), had high detection rates (56/60, P = 0.134, and 59/60, P = 1.000), and showed ideal specificity compared with SW (CDC). In addition, a cocktail of primer-probe sets targeted to the HA and NA genes displayed higher detection sensitivity than primer-probe sets targeting HA or NA alone, indicating that for practical applications, a combination of primer-probes targeting HA and NA genes is the best option for the detection of pandemic influenza A/H1N1 2009 virus.
authors.affiliation: State Key Laboratory for Molecular Virology and Genetic Engineering, Institute of Pathogen Biology (IPB), Chinese Academy of Medical Sciences and Peking Union Medical College (CAMS and PUMC), Beijing 100730, China.
authors.forename: Yaowu
authors.initials: Y
authors.lastname: Yang
authors.forename: Fang
authors.initials: F
authors.lastname: Huang
authors.forename: Richard
authors.initials: R
authors.lastname: Gonzalez
authors.forename: Wei
authors.initials: W
authors.lastname: Wang
authors.forename: Guilan
authors.initials: G
authors.lastname: Lu
authors.forename: Yongjun
authors.initials: Y
authors.lastname: Li
authors.forename: Guy
authors.initials: G
authors.lastname: Vernet
authors.forename: Qi
authors.initials: Q
authors.lastname: Jin
authors.forename: Jianwei
authors.initials: J
authors.lastname: Wang
mesh_terms: D017931:DNA Primers / Q000235:genetics*; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000235:genetics; D006801:Humans; D053118:Influenza A Virus, H1N1 Subtype / Q000235:genetics / Q000302:isolation & purification*; D007251:Influenza, Human / Q000175:diagnosis* / Q000821:virology*; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D009439:Neuraminidase / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D014764:Viral Proteins / Q000235:genetics; D014773:Virology / Q000379:methods*
chemical_list: D017931:DNA Primers; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D014764:Viral Proteins; C487630:NA protein, influenza A virus; D009439:Neuraminidase