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.

1. Assemble eluted DNA and/or RNA, PCR Reaction Mix, nuclease-free water as a negative control, and pooled standards for positive amplification control. NOTE: Up to 48 samples can be run on an amplification plate including controls (include at least one negative extraction control for each extraction plate from which samples are to be pulled). 2. Print out a sample map. Have a second person check that the map and sample layout match. 3 . In a 1.5 ml tube, add the following to prepare the preamp master mix. 4. Perform the preamp in a standard 96-well plate using the left side of the plate only (columns 1-6). 5. Add 8.5 µl of preamp master mix and 5.5 µl of DNA/RNA sample to each well. 6 . After combining all reagents (samples, positive controls and negative controls with Preamp mix), thoroughly seal the standard 96-well plate with a clear adhesive seal. Remove any excess seal using a razor blade to prevent formation of seal gaps during cycling. Centrifuge the sealed plate for 20 sec using a PCR plate spinner. Check to ensure all reagents are combined in the bottom of the plate wells. 7. Run the preamp assay on a conventional thermocycler under the following conditions: 15 min at 50 °C; 1 min at 95 °C; 20 cycles of 15 sec at 95 °C, then 2 min at 60 °C; 99.9 °C for 10 min; hold at 4 °C. Program these conditions prior to starting. 8. Turn on the conventional thermocycler, select the preamp cycling program and start the program. Place the 96-well plate in the thermocycler only when the bottom portion of the thermocycler (not the cover) reaches the temperature required for cDNA production (50 °C) by monitoring the temperature reading on the screen. Prior to this, keep the plate cold to minimize the risk of producing false positive results. 9. Once the preamp run is complete, verify that the plate has remained sealed. 10 . Proceed immediately to step 4.1. To store this plate overnight, perform the dilution as described in steps 4.2 to 4.5, except instead of loosely covering the plate, seal the plate with a clear adhesive seal and place inside a zipper-lock bag, stored at 4 °C.