Description: Background: Existing standard non-molecular diagnostic methods such as viral culture andimmunofluorescent (DFA) are time-consuming, labor intensive or limited sensitivity. Severalmultiplex molecular assays are costly. Therefore, there is a need for the development of arapid and sensitive diagnosis of respiratory viral pathogens. Methods: A GeXP-based multiplex RT-PCR assay (GeXP assay) was developed to detectsimultaneously sixteen different respiratory virus types/subtypes. Seventeen sets of chimericprimers were used to initiate the RT-PCR, and one pair of universal primers was used for thesubsequent cycles of the RT-PCR. The specificity of the GeXP assay was examined withpositive controls for each virus type/subtype. The sensitivity was evaluated by performing theassay on serial ten-fold dilutions of in vitro-transcribed RNA of all RNA viruses and theplasmids containing the Adv and HBoV target sequence. GeXP assay was further evaluatedusing 126 clinical specimens and compared with Luminex xTAG RVP Fast assay. Results: The GeXP assay achieved a sensitivity of 20-200 copies for a single virus and 1000 copieswhen all of the 16 pre-mixed viral targets were present. Analyses of 126 clinical specimensusing the GeXP assay demonstrated that GeXP assay and the RVP Fast assay were incomplete agreement for 109/126 (88.51 %) of the specimens. GeXP assay was more sensitivethan the RVP Fast assay for the detection of HRV and PIV3, and slightly less sensitive for thedetection of HMPV, Adv, RSVB and HBoV. The whole process of the GeXP assay for thedetection of 12 samples was completed within 2.5 hours. Conclusions: In conclusion, the GeXP assay is a rapid, cost-effective, sensitive, specific and highthroughput method for the detection of respiratory virus infections.

Description: Analytical Chemistry DOI: 10.1021/ac302853y