Abstract
BackgroundExisting standard non-molecular diagnostic methods such as viral culture and immunofluorescent (DFA) are time-consuming, labor intensive or limited sensitivity. Several multiplex molecular assays are costly. Therefore, there is a need for the development of a rapid and sensitive diagnosis of respiratory viral pathogens.MethodsA GeXP-based multiplex RT-PCR assay (GeXP assay) was developed to detect simultaneously sixteen different respiratory virus types/subtypes. Seventeen sets of chimeric primers were used to initiate the RT-PCR, and one pair of universal primers was used for the subsequent cycles of the RT-PCR. The specificity of the GeXP assay was examined with positive controls for each virus type/subtype. The sensitivity was evaluated by performing the assay on serial ten-fold dilutions of in vitro-transcribed RNA of all RNA viruses and the plasmids containing the Adv and HBoV target sequence. GeXP assay was further evaluated using 126 clinical specimens and compared with Luminex xTAG RVP Fast assay.ResultsThe 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. Analyses of 126 clinical specimens using the GeXP assay demonstrated that GeXP assay and the RVP Fast assay were in complete agreement for 109/126 (88.51%) of the specimens. 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 whole process of the GeXP assay for the detection of 12 samples was completed within 2.5 hours.ConclusionsIn conclusion, the GeXP assay is a rapid, cost-effective, sensitive, specific and high throughput method for the detection of respiratory virus infections.
Highlights
Existing standard non-molecular diagnostic methods such as viral culture and immunofluorescent (DFA) are time-consuming, labor intensive or limited sensitivity
For the viral targets that used clinical specimens as positive controls, including human bocavirus (HBoV), CoV HKU1, and CoV NL63, a single PCR product was detected in addition to the internal control peak
The results showed that the GeXP assay was more sensitive than the RVP Fast assay for the detection of human rhinovirus (HRV) and parainfluenza virus type 3 (PIV3), and less sensitive for the detection of human metapneumovirus (HMPV), Adv, respiratory syncytial virus B (RSVB) and HBoV
Summary
Existing standard non-molecular diagnostic methods such as viral culture and immunofluorescent (DFA) are time-consuming, labor intensive or limited sensitivity. There is a need for the development of a rapid and sensitive diagnosis of respiratory viral pathogens. The traditional assays used to diagnose respiratory tract viruses are viral culture and immunofluorescent staining. Viral culture remains the gold standard for the diagnosis of respiratory viruses because of its broad spectrum and high specificity. Viral culture is time-consuming and has a low sensitivity for the detection of some respiratory viruses that have fastidious growth requirements [5,6]. With the development of rapid molecular techniques, molecular assays, especially in a multiplex format, have been accepted as tests of choice for broad spectrum detection of respiratory viruses. All of them are based on a liquid-phase bead-based array technology to implement the detection, which has increased the cost and the implementation time of the whole assays
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