Development And Application Of One Tube Nested Real Time PCR For Detection Of Respiratory Viruses

Part one Significance of multiplex real time polymerase chain reaction technique in detection of respiratory syncytial virus and adenovirus:a meta-analysisObjective:To systematically review the diagnostic accuracies of multiplex real time polymerase chain reaction?mRT-qPCR?technique for detection of respiratory syncytial virus?RSV?and adenovirus?ADV?.Methods:PubMed,EMBASE,Cochrane,Wanfang and CNKI databases were searched from January1 2010 to January1 2018,to collect reports on mRT-qPCR for detection of common respiratory viruses.Then two authors independently exacted the data and assessed the risk of bias of included studies by using the QUADAS-2 tool.Meta-disc 1.4 was used to analyze the data.Deek test was used to assessed the publication bias.Results:Ten articles with 2528 cases were eligible for analysis.The results of meta-analysis showed that,the pooled sensitivity?Sen?,specificity?Spe?and area under SROC curve?AUC?for detecting RSV were 0.87?95%CI:0.83-0.90?,0.98?95%CI:0.97-0.98?and 1.00.The pooled Sen,Spe and AUC of mRT-qPCR for detecting ADV were 0.64?95%CI:0.56-0.71?,0.99?95%CI:0.98-0.99?and 0.99.Deeks test indicated that no publication bias was found.Conclusions:The sensitivity of mRT-qPCR in RSV and ADV detection is still to be improved,but the overall detection ability is good which deserves to be recommended for clinical use.Part two Development and appllication of one tube nested real time RT-PCR assay for detection of respiratory syncytial virusObjective:Respiratory syncytial virus?RSV?causes serious respiratory tract infection worldwide.The relatively low RSV load makes it difficult to detect in frail,elderly,and severely immune-compromised patients.Therefore,it is important to be able to detect RSV with high sensitivity and accuracy.Methods:In the present study,we developed a locked nucleic acid?LNA?-based one tube nested real time RT-PCR?OTNRT-PCR?assay with the advantages of extremely high sensitivity,facile operability,and less likelihood of cross-contamination.Sensitivity evaluation was performed using RSV strain,specific evaluation was performed using common respiratory viruses,and 616 clinical specimens were used for evaluation of clinical performance.The sensitivity,specificity,and clinical performance of the OTNRT-PCR assay were compared in parallel with a conventional TaqMan probe-based real-time PCR?RT-qPCR?assay and a traditional two-step nested PCR?N-PCR?assay.Results:The limit of detection of the OTNRT-PCR assay was1.02×10^-1 TCID50/ml,equivalent to the N-PCR assay,and 25-fold lower than the RT-qPCR assay.Of 616 clinical specimens tested,143 RSV-negative samples by RT-qPCR were confirmed as positive by sequencing the OTNRT-PCR products.Conclusions:We therefore conclude that OTNRT-PCR is more sensitive than RT-qPCR for the detection of RSV in clinical samples.Part three Development and application of a panel of multiplex one tube nested real time PCR assay for simultaneous detection of fourteen respiratory virusesObjective:Multiplex real time PCR?mRT-qPCR?assay is commonly used to detect respiratory viruses,however,the sensitivity is limited for most reports.Locked nucleic acid?LNA?-based multiplex one tube nested real time PCR?mOTNRT-PCR?assays were developed for simultaneous detection of respiratory syncytial virus?RSV?,human rhinovirus?HRV?and human metapneumovirus?HMPV?at the first step and later extended for detection of11 other respiratory viruses.Methods:The sensitivity and reproducibility of mOTNRT-PCR were evaluated using plasmid standards.The specific evaluation was performed using common respiratory viruses.The clinical performance of mOTNRT-PCR panel was further evaluated with clinical specimens and compared with individual real time PCR?RT-qPCR?assay.Results:The analytical sensitivities of mOTNRT-PCR for detection of RSV,HRV and HMPV were 5 copies/reaction.The analytical sensitivities of mOTNRT-PCR panel ranged from 2-20 copies/reaction,and no cross-reaction with common respiratory viruses was observed.Totally 33 clinical samples detected by mOTNRT-PCR for detection of RSV,HRV and HMPV were missed by RT-qPCR and confirmed true positive by sequencing of nested PCR?N-PCR?products and 35 clinical samples detected by mOTNRT-PCR assay panel were missed by RT-qPCR and confirmed true positive by sequencing of N-PCR products.Conclusions:The mOTNRT-PCR assay panel provides a more sensitive than RT-qPCR and high-throughput method for the detection of 14 respiratory viruses.