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

Mohammad Rubayet Hasan; Hassan Al Mana; Virginia Young; Patrick Tang; Eva Thomas; Rusung Tan; Peter Tilley

Real-time PCR: Primers and probes for sixteen PCR assays were either obtained from 141 previously published assays or newly designed, in this study by using the Primer Express 142 software v3.0.1 (Life Technologies) ( Table 1) . The in silico specificity of the amplicon sequence 143 for the target pathogen was confirmed by nucleotide blast search against the NCBI non- The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/455568 doi: bioRxiv preprint QuantiTect RT Mix (Qiagen, USA). Ten percent extra volume was added to the required volume 185 for all components, to account for pipetting error. Next, using an electronic dispensing pipette 186 (Eppendorf, USA), 17.5 µl of sample mix was dispensed into the 16-wells of the 96-well PCR plate, in the two adjacent columns containing the primers and probes as described above and in enterovirus, multiple primer and probe sets were tested for superior performance. Because of the 218 cross-reactivity of enterovirus and rhinovirus primers and probes with their respective targets, an 219 additional PCR reaction was added for enterovirus 68 in order to correctly assign results for 220 enteroviruses and rhinoviruses. PCR assays were then tested individually using PCR, DFA or 221 culture confirmed specimens or ATCC reference strains of bacteria and viruses (n=397).