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.

The Final Results worksheet in the supplemental file shows representative quantitative results for 10 clinical diagnostic samples and 4 controls. The clinical samples are a subset of the types of samples routinely tested including nasal/oropharyngeal swabs, lung tissue, and fecal samples. One (equine) fecal sample in this set shows a failed MS2 internal control, which indicates the presence of inhibitors in the sample. This is typically managed by dilution of the eluted sample and/or re-extraction. As is the case here, the negative amplification control should be negative for all targets, and the negative extraction control should only contain the internal control. In the clinical set, samples produced Ct values for betacoronavirus, Bordetella bronchiseptica, canine distemper virus A, canine parainfluenza virus, canine pneumovirus, and influenza A. Table 1 . Schematic of the amplification plate and target locations. The plates used for nanoscale real-time PCR testing on this platform are microscope slide-sized and are arranged in 48 subarrays of 64 through-holes, with a total of 3,072 through-holes for individual reactions. One subarray is shown here, with 18 targets in triplicate. Each sample is added to one subarray by the liquid handler. Plates are coated with hydrophilic and hydrophobic compounds to retain reagents in through-holes via surface tension. The stainless steel chip is "photolithographically patterned and wet-etched to form a rectilinear array of 3,072 micro-machined, 320 µm diameter holes of 33 nl each" Table 2 . Plate transfer map. A color-coded plate transfer map for using a fixed 8-channel pipette to transfer from the 96-well preamp plate to the 384-well plate is shown. Alternately, an adjustable pipette may be used. The same procedure is performed each time regardless of how many samples are in the plate.