High-throughput Detection of Respiratory Pathogens in Animal Specimens by Nanoscale PCR

Goodman, Laura B.; Anderson, Renee R.; Slater, Marcia; Ortenberg, Elen; Renshaw, Randall W.; Chilson, Brittany D.; Laverack, Melissa A.; Beeby, John S.; Dubovi, Edward J.; Glaser, Amy L.

Due to the very small amount of sample loaded onto the amplification plate (33 nl), pre-amplification is required. In the optimization of this protocol (not shown), we compared a number of preamplification parameters including the master mix, addition of random primers, number of cycles, annealing time, and dilution prior to amplification. Each target had its own optimal conditions, and those described here represent those that yielded the best limit of detection overall for our panel. This panel covers a wide range of pathogenic targets and sample types that are encountered in routine veterinary diagnostic testing, but modifications to the preamplification procedures may be necessary for different panels. The manufacturer-optimized amplification conditions are based on a standard probe-based real-time PCR protocol with a 10 min at 95 °C enzyme activation followed by denaturation at 95 °C and annealing/extension at 60 °C. The primers and probes are pre-printed on the plates also using manufacturer-optimized conditions and therefore do not require titration. Combining the reverse-transcription and preamplification steps for all samples (regardless of the type of target) was necessary in order to maintain efficiency in the workflow. Having all samples reverse transcribed is also beneficial for maximizing sensitivity. Furthermore, use of a master mix for the preamp that is optimized for minimizing inhibitors allows versatility in combining different sample types on the same plate.