title: Detection of the pandemic H1N1/2009 influenza A virus by a highly sensitive quantitative real-time reverse-transcription polymerase chain reaction assay.
abstract: A quantitative real time reverse-transcription polymerase chain reaction (qRT-PCR) assay with specific primers recommended by the World Health Organization (WHO) has been widely used successfully for detection and monitoring of the pandemic H1N1/2009 influenza A virus. In this study, we report the design and characterization of a novel set of primers to be used in a qRT-PCR assay for detecting the pandemic H1N1/2009 virus. The newly designed primers target three regions that are highly conserved among the hemagglutinin (HA) genes of the pandemic H1N1/2009 viruses and are different from those targeted by the WHO-recommended primers. The qRT-PCR assays with the newly designed primers are highly specific, and as specific as the WHO-recommended primers for detecting pandemic H1N1/2009 viruses and other influenza viruses including influenza B viruses and influenza A viruses of human, swine, and raccoon dog origin. Furthermore, the qRT-PCR assays with the newly designed primers appeared to be at least 10-fold more sensitive than those with the WHO-recommended primers as the detection limits of the assays with our primers and the WHO-recommended primers were 2.5 and 25 copies of target RNA per reaction, respectively. When tested with 83 clinical samples, 32 were detected to be positive using the qRT-PCR assays with our designed primers, while only 25 were positive by the assays with the WHO-recommended primers. These results suggest that the qRT-PCR system with the newly designed primers represent a highly sensitive assay for diagnosis of the pandemic H1N1/2009 virus infection.
authors: Institute of Virology, School of Life Sciences, Nanjing University, Nanjing 210093, Jiangsu, China.
authors: Zhu
authors: Z
authors: Yang
authors: Guoliang
authors: G
authors: Mao
authors: Yujun
authors: Y
authors: Liu
authors: Yuan-Chuan
authors: YC
authors: Chen
authors: Chengjing
authors: C
authors: Liu
authors: Jun
authors: J
authors: Luo
authors: Xihan
authors: X
authors: Li
authors: Ke
authors: K
authors: Zen
authors: Yanjun
authors: Y
authors: Pang
authors: Jianguo
authors: J
authors: Wu
authors: Fenyong
authors: F
authors: Liu
mesh_terms: D002681:China / Q000453:epidemiology; D017931:DNA Primers / Q000235:genetics; D006801:Humans; D053118:Influenza A Virus, H1N1 Subtype / Q000235:genetics / Q000302:isolation & purification*; D009981:Influenza B virus / Q000235:genetics / Q000302:isolation & purification; D007251:Influenza, Human / Q000175:diagnosis / Q000453:epidemiology / Q000821:virology*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D014764:Viral Proteins / Q000235:genetics
chemical_list: D017931:DNA Primers; D014764:Viral Proteins
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