Establishment and application of Real-time quantitative PCR for human Metapneumovirus detection

Abstract

Objective To establish and evaluate a Metapneumovirus real-time quantitative reverse transcription polymerase chain reaction (RT-PCR), detect clinical specimens and explore the clinical prevalence characteristics of Metapneumovirus-infected children. Methods The primers and probes which targeted the conserved genes F were designed, RNA standards were prepared to establish a standard curve, the sensitivity, specificity and reproducibility had been tested. 222 lower respiratory clinical specimens were collected from children in Lanzhou area with ARI. Metapneumovirus and co-infection viruses were detected simultaneously; further Metapneumovirus related epidemiology was studied. Results Metapneumovirus linear detection range was 10-108 copies/μl, the lowest detection limit was 10 copies/μl, the correlation coefficient was 1, the amplification efficiency was 91.62%, the CV of Ct value was less than 2%. Take conventional PCR product sequence results as reference, real-time quantitative RT-PCR sensitivity and specificity were 100% and 96.17%. Metapneumovirus detection rate were 9.46%, 13 cases for boys (5.86%), 8 case for girls (3.60%). The detection rate of spring, summer, autumn and winter were 15.71%, 0%, 5.08%, 11.11%. There were no significant differences between the Metapneumovirus viral load and mixed infection or the types of disease, clinical symptoms. Conclusions Metapneumovirus real-time quantitative RT-PCR has been confirmed as a sensitive and specific method. Metapneumovirus was an important agent of children respiratory tract infection in Lanzhou region.We should take the long-term systematic surveillance seriously. Key words: Real-time quantitative reverse transcription PCR; Metapneumovirus