Development And Application Of Nucleic Acid Assays For Detection Of Human Bocavirus And Human Metapneumovirus

Among children,acute respiratory tract infections(ARTIs)are a major cause of morbidity and mortality worldwide.In the past few decades, due to the more advanced instruments application and more sensitive detection methods, more respiratory viruses were found such as Human Bocavirus and Human Metapneumovirus.However,there are also some respiratory tract illnesses in childern that the pathogens can not be identified from respiratory samples.The major content and reasults of our research as follows:1. To establish two conventional PCR/RT-PCR assays separately for Human Bocavirus(HBoV) and Human Metapneumovirus(hMPV),and to detect HBoV and hMPV from nasopharyngeal samples of children with ARTI. The sensitivity of the established conventional PCR/RT-PCR for HBoV NP1 gene and hMPV M gene were 250 DNA copies/reaction and 500 DNA copies/reaction respectively with a better specificity.2. The specific primers and Taqman probes used for real-time Fluorescent Quantitative PCR/RT-PCR were designed respectively according to the conservative sequence.Subsequently, experiments were undertaken to assess diagnostic criteria such as specificity, sensitivity and reproducibility. The sensitivity of real-time Fluorescent Quantitative PCR assay for HBoV was 10 DNA copies/reaction and the sensitivity of real-time Fluorescent Quantitative RT-PCR assay for hMPV was 4.95 DNA copies/reaction,both of the realts with the better specificity and reproducibility.3. A total of 76 nasopharyngeal swab specimens derived from Shenyang were screened for the presence of HBoV and hMPV using conventional PCR/RT-PCR and real-time Fluorescent Quantitative PCR/RT-PCR respectively.5 specimens were identified positive for HBoV by real-time Fluorescent Quantitative PCR assay,and 3 positive specimens screened by conventional PCR,and the specimens identified by conventional PCR were contained in the specimens identified by real-time Fluorescent Quantitative PCR assay.As for the hMPV,7 positive specimens were detected by real-time Fluorescent Quantitative RT-PCR with a higher dectection rate than that identified by conventional RT-PCR assay(5/76),and the result of the conventional PCR were according with that of real-time Fluorescent Quantitative RT-PCR assay. ConclusionsThe nucleic acid assays for detection of HBoV and hMPV have been developed and were preliminary proved to have a better sensitivity,specificity,reproducibility and these assays maybe applied for epidemical surveillance and clinical diagnosis of HBoV and HMPV.The comparison results of convential PCR/RT-PCR and realtime Fluorescent Quantitative PCR/RT-PCR showed that realtime Fluorescent Quantitative PCR/RT-PCR is easy to operate,faster and more sensitive than convertial PCR/RT-PCR.