Detection And Analysis Of HBoV And HCoV-229E In Children With Acute Respiratory Tract Infections By FQ-PCR

ObjectiveTo establish two real-time Fluorescent Quantitative PCR(FQ-PCR) assays separately for Human Bocavirus(HBoV) and Human Coronavirus 229E(HCoV-229E), and to detect HBoV and HCoV-229E from nasopharyngeal samples of children with acute respiratory tract infections(ARTI).MethodsThe specific primers and Taqman probes were designed targeting the HBoV NP1 gene and HCoV-229E M gene respectively. The aimed fragments of NP1 gene and HCoV-229E M gene were amplified with PCR and ligated into pGEMT-Easy vector for standards . Two assays for detection of NP1 gene and M gene using FQ-PCR were established separately after determination of the sensitivity and specificity of the tests, and 195 clinical specimens from children with ARTI were subsequently tested. Two general PCRs also developed and used to detect HBoV and HCoV-229E in the 195 clinical specimens.Results1. The sensitivity of the established FQ-PCR for HBoV NP1 gene was 101copies/μl and the CVs of inter-assay and intra-assay were 0.13% and 0.14% respectively.2. By using FQ-PCR, twelve of 195 clinical specimens(6.2%) were tested positive for HBoV. The results of the general PCR were according with that of FQ-PCR.3. All of 12 chidren with HBoV positive detection are aged less than 4 years. The HBoV positive rate in lower respiratory tract infections group was 15.6%(10/64),significantly higher than that of 1.5% (2/131) in upper respiratory tract infections group(X2=12.45,P<0.005).4. The sensitivity of the established FQ-PCR for HCoV-229E M gene was 101copies/μl and the CVs of inter-assay and intra-assay were 0.4% and 0.7% respectively.5. By using FQ-PCR, two of 195 clinical specimens(1.0%) were tested positive for HCoV-229E. The results of the general PCR were according with that of FQ-PCR .6. Two positive HCoV-229E samples were from children with lower respiratory tract infections group.Conclusions1. Two FQ-PCRs for HBoV and for HCoV-229E respectively were established with satisfactory sensitivity, specificity and reproducibility. These assays may be applied in clinical diagnosis and epidemical surveillance of HBoV and HCoV-229E infections.2. HBoV and HCoV-229E were prevalent in children with ARTI in Fuzhou area in winter-spring seasons. HBoV maily cause ARTI in infants and children aged below 4 years...