A novel real-time PCR assay panel for detection of common respiratory pathogens in a convenient, strip-tube array format

Abstract

Commercial multiplex assays, built on different chemistries and platforms are widely available for simultaneous detection of pathogens that cause respiratory infections. However, these tests are often difficult to implement in a resource limited setting because of high cost. In this study, we developed and validated a method for simultaneous testing of common respiratory pathogens (Respanel) by real-time PCR in a convenient, strip-tube array format. Primers and probes for sixteen PCR assays were selected from the literature or newly designed. Following optimization of individual PCR assays, strip-tube arrays were prepared by dispensing primer-probe mixes (PPM) into two sets of 8-tube strips. Nucleic acid extracts from specimens were mixed with PCR master mix, and dispensed column-wise into 2 × 8-wells of a 96-well plate. PPMs from strip-tubes were then added to the wells using a multichannel pipette for real-time PCR. Individual PCR assays were optimized using previously known specimens (n = 394) with 91%-100% concordance with culture, DFA or PCR results. Respanel was then tested in a routine manner at two different sites using specimens (n = 147) previously tested by Qiagen Resplex I&II or Fast-Track Diagnostics Respiratory Pathogens 21 assays. The sensitivity, specificity and accuracy of Respanel were 94%, 95% and 95%, respectively, against Resplex and 88%, 100% and 99%, respectively, against FTDRP21. Respanel detected more pathogens (p < 0.05) than Resplex but the rate of pathogen detection was not significantly different from FTDRP21. Respanel is a convenient and inexpensive assay that is more sensitive than Resplex and comparable to FTDRP21 for the detection of common respiratory pathogens.

Keywords: Commercial multiplex assays; Laboratory-developed tests; Real-time PCR; Respiratory pathogens; Validation.