Detection And Analysis Of HCoV-NL63 And HCoV HKU1 In Children With Acute Respiratory Tract Infections By Fq-pcr In Fuzhou

ObjectiveTo establish a real-time fluorescent quantitative PCR(FQ-PCR)assay and to detect the human coronavirus NL63(HCoV-NL63) and human coronavirus HKU1 (HCoV-HKU1) from nasopharyngeal samples of children with acute respiratory tract infec-tions (ARTI) in Fuzhou.MethodsThe specific primers and Tap-man probes were designed targeting the HCoV-NL63 1a gene and HCoV-HKU1 pol gene. The two aimed fragment were amplified with PCR and ligated into a PMD18-T Easy vector for standards.One hundred and fifty-one clinical specimens were subsequently tested after determination of the sensitivity and specificity of the established real-time PCR. .Amplification and sequencing of N gene and E gene of HCoV-NL63 from the positive specimens were performed for homological analysis and phylogenetic analysis.Results 1. The sensitivity of the established FQ-PCR for HCoV-NL63 was 101 copies/μl～109copies /μl and the CVs of inter-assay and intra-assay were 0.9% and 1.6% respectively. By using the FQ-PCR, two of 151 clinical specimens(1.3%) were tested positive for HCoV-NL63 in Fuzhou from winter 2008 to spring 2009.2.The nucleotide sequence and the amino acid sequence of HCoV-NL63 nucleocapsidprotein are 99% and 97% homology with those of the reference strains in the GenBank respectively. There are three amino acid of nucleocapsidprotein differ from the reference strains in the GenBank. The nucleotide sequence of envelope protein is 99% homology with the wild strains while the amino acid sequence of envelope protein is 100% homology with the reference strains.3.The sensitivity of the established FQ-PCR for HCoV-HKU1 was 102 copies/μl～ 109copies /μl and the CVs of inter-assay and intra-assay were 1.2% and 0.5% respectively. By using the FQ-PCR, none of clinical s was tested positive for HCoV-HKU1 in Fuzhou from winter 2008 to spring 2009. Nevertheless,one specimen stored in our lab proved HCoV-HKU1 positive by ordinary RT-PCR before also showed positive using the FQ-PCR for HCoV-HKU1.Conclusions1.The real-time fluorescent quantitative PCR assays was successfully established to detect HCoV- NL63 and HCoV-HKU1.2.The N gene and E gene of HCoV-NL63 deteted from ARTI children in Fuzhou both have high homology with the wild strains in GenBank.3.HCoV-NL63 was prevalent with the lower incidence of 1.3% in children with ARTI in Fuzhou area in 2008-2009 winter-spring seasonas. That none of the 151 children was found for HCoV-HKU1 indicate even much lower incidence for the virus in children with ARTI in this area.