Development Of Real Time RT-PCR Assays For Four Respiratory Viruses And Their Clinical Applications

Vial infections of the acute respiratory tract illness(RTIs) are responsible for significant mortality and morbidity worldwide.And the costs attributable to RTIs are an important burden on national healthcare budgets.So it plays a very important role to detect and surveillance the pathogens of respiratory tract infection.Despite extensive studies in the past decades that have identified a number of etiologic agents,including Influenza A and B,RSV,Parainfluenza 1,2 and 3, Adenovirus,rhinoviruses and coronaviruses(HCoV-OC43,HcoV-229E), approximately 30%of all cases cannot be attributed to these agents,suggesting that additional respiratory pathogens are exist.In fact,several new viruses have been identified in patients with LRTIs since 2001.HCoV-NL63,HCoV-HKU1 and HMPV are recently described respiratory viruses that have been reported worldwide,but a few in China.Diagnostic tests for HCoVs and HMPV are not frequently used in the routine setting in China.It is likely that,as a result,the precise role that HCoVs and HMPV play in RTIs is greatly underestimated.Hence,it is very important to develop a fast,sensitive and specific method for the research of characteristics,evolution, prevalence,pathogenesis,clinical manifestation and defensive effect of above-mentioned viruses.Much of the early diagnostic work on HCoVs or HMPV relied on cumbersome and insensitive methods such as serologic analysis,virus culture,and antigen detection.The subsequent development of sensitive reverse-transcription polymerase chain reaction(RT-PCR) assays has permitted a wider study of these viruses, increasing the number of reports possibly linking HCoV infection to more-severe lower respiratory tract infections.Rapid and reliable diagnosis of HCoVs or HMPV infections therefore becomes indispensable in a routine clinical setting.Real time RT-PCR is firmly established as a mainstream research technology in recent year.A major advantage of real-time RT-PCR is that amplification and analysis are completed in a closed system,making it less time-consuming than(nested) RT-PCR methods, which still require post-PCR analysis.The risk of contamination,which can confound conventional(nested) RT-PCR protocols,is markedly reduced.According to the representative sequences of three kinds of HCoVs and HMPV, we designed specific primers and probes to develop four real-time RT-PCR assays for detection them.158 nasopharyngeal swab specimens of fever or pneumonia patients were screened for the presence of above-mentioned viruses by using real time RT-PCR and assessed in parallel by the conventional RT-PCR assays.1.Development of real time RT-PCR assays for four kinds of respiratory viruses (HCoV-229E,HCoV-NL63,HCoV-HKU1 and HMPV)Specific primers and probes were designed basing on representative sequences of above four kinds of viruses,and then the corresponding gene was cloned to downstream of pET-9a vector T7 promoter and in vitro transcribed as positive template.Absolute viral load measurement in samples was achieved through above cRNA standards of in-house HCoVs(HCoV-229E,HCoV-NL63,HCoV-HKU1) and HMPV ranging from 10 to 10~7 copies per reaction mixture for the generation of a standard curve.Subsequently,experiments were undertaken to assess diagnostic criteria such as specificity,sensitivity and reproducibility.The analytical detection limit of all the four kinds of real-time RT-PCR assays was 10 copies cRNA.In order to exclude nonspecific reactions,other three kinds of cRNA were subjected to amplification in the real-time RT-PCR for current assay and no nonspecific amplification was obtained in each assay.To assess the intra-assay reproducibility of every real-time RT-PCR assay,tenfold dilution of the cRNA standards for five different amount copies per reaction mixture were analyzed in five replicates per run. Coefficient of variation was lower than 5%for all real-time RT-PCR assays. 2.The clinical applications of four kinds of real-time RT PCR assaysTo evaluate the clinical application of the real-time RT-PCR assays for detection above four kinds of viruses,we analyzed 158 nasopharyngeal swab specimens from patients hospitalized with pneumonia,outpatients with influenza-like illness,and asymptomatic control patients which were collected from Beijing during October and November,2007.In the 158 specimens,real-time RT-PCR in nasopharyngeal aspirates revealed HCoV or HMPV infection as follow:103 HCoV-229E(65.2%),6 HCoV-NL63(3.8%), 5 HCoV-HKU1(3.2%) and 31 HMPV(19.6%0;In contract,the presence of HCoV-229E,HCoV-NL63,HCoV-HKU1 and HMPV were 62.7%(99/158),1.9%(3/158),1.9% (3/158),13.9%(22/158) respectively when the specimen were examined with traditional RT-PCR.Overall sensitivity for detecting virus was higher for real-time RT PCR assay compared to conventional RT-PCR assays.All samples positive by conventional RT-PCR were confirmed by sequencing,except one of the HCoV-229E.In summary,We have developed real-time RT PCR assays that can detect and identify different four common(HCoV-229E) or emerging(HMPV,HCoV-NL63, HcoV-HKU1) human respiratory viruses in 5 hour;Tested 158 NP specimens by real-time RT PCR(or conventional RT-PCR) assay-103(or 99) were positive for HCoV-229E,6(or 3) were positive for HCoV-NL63,5(or 3) were positive for HCoV-HKU1,31(or 22) were positive for HCoV-HMPV,respectively.All positive specimens by RT PCR were confirmed by sequencing.Clinical importance of these infections(HMPV,HCoV-229E,HCoV-NL63,HcoV-HKU1) in patients need to be further determined.The real-time RT PCR assays described here may increase our understanding of epidemiology of respiratory virus infections,and should increase the detection rate for viral etiological agent's identification in the clinical specimens for hospitalized individuals with RTI or community RTI outbreaks.