Development of real-time RT-PCR for detection of human metapneumovirus and genetic analysis of circulating strains (2009-2011) in Pune, India.
Human metapneumovirus (HMPV) is an important respiratory virus implicated in respiratory infections. The purpose of this study was to develop a one-step real-time RT-PCR assay that can detect all four lineages of HMPV and to identify the HMPV lineages circulating in Pune, India. Conserved regions of the nucleoprotein gene were used to design real-time primers and a probe. A total of 224 clinical samples that were positive for different respiratory viruses (including 51 samples that were positive for HMPV) were tested using the real time RT-PCR assay, and the specificity of the assay was observed to be 100 %. Using in vitro-synthesized RNA, the sensitivity of the assay was ascertained to be 100 copies of the target gene per reaction. Phylogenetic analysis of the nucleoprotein (N) and attachment glycoprotein (G) genes confirmed that this assay detected all lineages of HMPV. A2, B1 and B2 strains were observed during the study period. Our assay is highly sensitive and specific for all known lineages of HMPV, making it a valuable tool for rapid detection of the virus. A2 and B2 were the predominant subtypes circulating in Pune, Western India.
Human Influenza Department, National Institute of Virology, 20-A, Dr. Ambedkar Road, Pune, 411001, India, mlchoudhary@gmail.com.
Manohar Lal
ML
Choudhary
Siddharth P
SP
Anand
Nupoor S
NS
Sonawane
Mandeep S
MS
Chadha
D016000:Cluster Analysis; D017931:DNA Primers / Q000235:genetics; D006801:Humans; D007194:India; D029121:Metapneumovirus / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D008969:Molecular Sequence Data; D018184:Paramyxoviridae Infections / Q000175:diagnosis* / Q000821:virology*; D010802:Phylogeny; D012367:RNA, Viral / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012141:Respiratory Tract Infections / Q000175:diagnosis / Q000821:virology; D012189:Retrospective Studies; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D014764:Viral Proteins / Q000235:genetics; D014773:Virology / Q000379:methods*
D017931:DNA Primers; D012367:RNA, Viral; D014764:Viral Proteins