Development Of Two Novel Multiplex RT-PCR Assays For Simultaneous Detection Of 16 Human Respiratory Virus Types/Subtypes Based On Capillary Electrophoresis

Background:Viral respiratory tract infections, which have a considerable morbidity and fatality rate, are common diseases that especially affect infants and the elderly. Existing standard non-molecular diagnostic methods such as viral culture and immunofluorescent (DFA) are time-consuming, labor intensive or limited sensitivity. Several commercial multiplex molecular assays are costly, which restricts the popularizing rate of these assays in the routine surveillance of respiratory virus infections in China. Therefore, there is a critical need for the development of a rapid, cost-effective, sensitive, specific and high throughput method for the detection of respiratory virus infections.Objective:In this study, two novel one-step multiplex RT-PCR assays based on GeXP system and Qiaxcel automatic capillary electrophoresis have been developed in single or two reactions to detect simultaneously sixteen common respiratory virus types/subtypes, containing influenza A virus, influenza B virus, seasonal influenza A H1N1 virus, parainfluenza virus type 1-3, human rhinovirus, human metapneumovirus, adenovirus, respiratory syncytial virus A, respiratory syncytial virus B, four coronavirus sybtypes (CoV HKU1, CoV NL63, CoV 229E, CoV OC43) and human bocavirus. The specificity and sensitivity of the two novel assays were examined, and the clinical performance of the two novel assays were evaluated in the detection of clinical specimens compared to commercial xTAG RVP Fast assay.Method:The temperature switch PCR (TSP) strategy was adopted to optimize the amplification parameters. Seventeen sets of chimeric primers were used to initiate the RT-PCR, one pair of universal primers was used for the subsequent cycles of the RT-PCR, and one pair of primers was internal control. All of the primers were contained in one reaction to implement the GeXP assay. For the Qiaxcel assay, nine pairs of chimeric primers were contained in tube 1 to detect nine respiratory viruses, and eight pairs of chimeric primers were contained in tube 2 to detectseven respiratory viruses. The specificity of the GeXP/Qiaxcel assaywas examined with positive controls for each virus type/subtype. The sensitivity was evaluated by performing the assay on serial ten-fold dilutions of viral RNA transcripts of all RNA viruses and the recombinant plasmids containing the Adv and HBoV target sequence. The same copies of each virus were then pre-mixed to evaluate the sensitivity when all of the 16 viral targets were present. GeXP assay was further evaluated using 126 clinical specimens and compared with xTAG RVP Fast assay. For the Qiaxcel assay, a total of 247 specimens were used to evaluate the two-tube assay and the results were compared with those obtained from the xTAG RVP Fast assay. The discordant results between the Qiaxcel assay and the RVP Fast assay were confirmed by sequencing or by the Seeplex RV15 ACE detection kit.Results:1. The GeXP assay achieved a sensitivity of 20-200 copies for a single virus and 1000 copies when all of the 16 pre-mixed viral targets were present. No cross-reactivity among the 16 respiratory virus types/subtypes was observed. Analyses of 126 clinical specimens demonstrated that GeXP assay was more sensitive than the RVP Fast assay for the detection of HRV and PIV3, and slightly less sensitive for the detection of HMPV, Adv, RSVB and HBoV. The accordance rates of GeXP assay and the RVP Fast assay for detection of sixteen viruses were above 92.86%, which demonstrated that the GeXP assay had comparable sensitivity and specificity to the commercially available RVP Fast assay.2. The Qiaxcel assay achieved a sensitivity of 20-200 copies per reaction individually for each virus type/subtype. The sensitivity was 2000 copies with 9 pre-mixed viral targets in tube 1 (only the amplicons of sH1N1 and CoV 229E were absent with dilutions of 200 copies), and 200 copies in tube 2 with 8 pre-mixed templates (only the amplicons of CoV NL63, HMPV and RSVB were absent with dilutions of 20 copies). The overall detection rate of the Qiaxcel assay for each virus was comparable to that of the RVP Fast assay (kappa>0.75). The accordance rates were above 95.14% except for HRV. The confirmed results by sequencing or by the Seeplex RV15 showed that Qiaxcel assay detected no false positives but six false negatives. All the accordance rates were above 98.79% compared to the confirmed results.Conclusions:We successfully developed two novel multiplex RT-PCR assays which were rapid, cost-effective, sensitive, specific and high throughput.for simultaneous detection of sixteen respiratory virus types/subtypes. The two novel assays may have a great potential for routine surveillance of respiratory virus infection in China to improve the capacity for emergency management.