Detection of human corona viruses non SARS in clinical specimens from patients with respiratory tract infection by use of real-time reverse-transcriptase polymerase chain reaction with taqman probes

Objective To develop a sensitive specific and rapid real-time quantitative polymerase chain reaction (PCR) assay using Taqman probe for detecting four common human coronavirus (HCoV) excluding SARS.To detect four coronaviruses in children with acute respiratory infection use the assay.Methods We designed and obtained specific pairs of primers and Taqman probes sequences to identify type-specific conserved regions of the four viruses.Simultaneous Detection of the four HCoV (non SARS) by using fluorescent real time PCR and common real time PCR respectively for 248 samples.The specificity,sensitivity and stability of the assay were evaluated.Results The detection ability for NL63,HKU1,229E by using the designed assays were the same as common RT-PCR,but for OC43 the assays demonstrates higher sensitivity compared with common RT-PCR.No cross-reaction with the other examined RNA viruses was observed.The detection limits were up to 10 copies/μl and this assay allowed quantitation of four viruses over a range of 101 to 108 RNA copies/μl.These results and the ability to detect virus in sample by RT-PCR demonstrate the higher sensitivity of the TaqMan assay compared with that of a conventional RT-PCR assay,viruses positive samples were all examed in December till May of next year.Conclusions The results showed that the assay had high sensitivity and specificity.TaqMan RT-PCR have proven useful in assisting scientists in respiratory tract and it is an effective way for clinical examination and surveillance of coronavirus. Key words: Coronavirus;  Revers trascriptase polymerase chain reactions;  Fluorescent antibody technique