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

The overall goal of this real-time PCR testing methodology is to detect both DNA and RNA based pathogens together in a high-throughput manner. Nucleic acid from clinical specimens will be pre-amplified with a common primer pool loaded onto an OpenArray plate and analyzed for the presence of pathogens. This method can help promote OneHealth surveillance efforts for infectious diseases.

Compared with traditional techniques, this method allows more samples to be tested for more targets without competing with each other, thereby improving testing efficiency. The method uses a customized nanoscale PCR plate. The primers and probes are printed on the edges of through holes and the samples are loaded using a liquid handler.

We will demonstrate the method on clinical specimens being tested for a panel of respiratory pathogens of horses and dogs. To begin this procedure, design the plate as described in the text protocol. The plate used here is configured with 18 targets in triplicate.

Add media to the swabs and tracheal washes so that there is at least one to two milliliters of liquid. Vortex the swab and DMEM vigorously in the tube. Then, use a pipette to transfer approximately 1 milliliter of media to a new tube.

Add 235 micro liters of lysis buffer and 175 micro liters of sample to each bead tube. Proceed with the manufacturer’s protocol to extract the total nucleic acid. Prepare for reverse transcription and pre-amplification of nucleic acid or preamp as described in the text protocol by first verifying the sample map.

Add 8.5 micro liters of preamp master mix and 5.5 micro liters of DNA/RNA sample to each well of a standard 96-well plate using the left side of the plate only. After combining the 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 seconds using a PCR plate spinner. Check to ensure all reagents are combined in the bottom of the plate wells. After turning on a 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 reaches the temperature required for cDNA production by monitoring the temperature reading on the screen. Once the preamp run is complete, verify that the plate has remained sealed. After donning double gloves and sleeve covers, remove the seal from the preamp plate.

Then, carefully remove and discard the outer gloves. Dilute the preamp products one to five by adding 56 micro liters of TE buffer to each well of columns one through six of the 96-well preamp plate and mixing by pipetting up and down. To do so, go only to the first stop of the pipette, aspirate from the bottom and dispense higher up but do not create bubbles.

Add TE buffer to all six columns regardless of unused wells. Prepare for the 384-well transfer to the amplification plate as described in the text protocol. Upon completion of the preparation steps, put on new well-fitted double gloves and sleeve covers.

Due to high evaporation rates while pipetting low volumes, it is critical to dispense master mix and sample efficiently and seal the plate immediately. Add 2.5 micro liters of amplification master mix to a 384-well plate. White plates are used here for demonstration purposes but black plates should be used to reduce eye strain.

Transfer 2.5 micro liters of the diluted preamp product to the 384-well plate according to the map in the text protocol. The colored dice shown here, illustrate the transfer sequence for a fixed channel pipette. Immediately cover the plate tightly with a foil seal.

Discard outer gloves and sleeve covers. Centrifuge the 384-well plate for 20 seconds in the PCR plate spinner. Do not remove the foil seal, but place the 384-well plate in the liquid handler.

Open the package containing an encased amplification plate and place it carefully in the liquid handler in the first slot with the serial number on the right, hold the case by the edges without touching the surface of the plate. Remove the foil seal from the 384-well plate inside the liquid handler once everything is in place. Click next and then check all the boxes as each step is completed.

Close the door to the liquid handler and immediately start the filling process. Stay next to the liquid handler and immediately proceed to the next step once the plate is filled. While the liquid handler is running, remove the protective film from the bottom of a case lid.

Leave the top film in place. Prime the red tape by pulling slightly to release it from the adhesive. Remove the loaded amplification plate from the liquid handler and gently place it in the plate press with the serial number on the right.

Place the lid on top of the plate with the notched end on the right and the adhesive on the bottom. Pull down on the plate press lever carefully closing it, the light will flash for 20 seconds. Do not touch the plate press during this time.

Once the light turns solid green, lift up the lever and carefully remove the sealed plate by holding it on the edges. While holding the sealed amplification plate vertically by the edges of the case, immediately insert the syringe tip into the loading port at the end of the case, then dispense the immersion fluid slowly in one gentle continuous motion to fill the space between the plate and the lid. Leave one small air bubble in the corner once the plate is immersed.

While continuing to hold the plate vertically by the edges, seal the loading port by inserting the plug into the port and twisting the plug clockwise, apply sufficient pressure until the handle breaks off. Clean the case with a lint-free wipe that has been thoroughly sprayed with ethanol. To dry the case, wipe the case downward with a clean wipe.

Bring the sealed plate to the amplification machine. Under the instrument tab, select instrument console. Select the amplification machine and then click on the open door button.

Place the amplification plate in the first slot of the plate adapter, checking that the adapter is correctly lined up with a one in the upper left corner. Orient the plate with the barcode facing up and toward the front of the instrument. After clicking the close door button, navigate to the home tab at the bottom of the screen.

Under the run menu, select OpenArray. Click get plate IDs. To begin the run, click start run.

Open a spreadsheet containing the results macro. In the exported data file, right click on the tab at the bottom of the screen and select move or copy. In the to book field, select results macro.

Then click move to end and check the box labeled create copy. Finally, click okay. Delete the old export tab and then rename the newly added tab as export.

Click view then macros, and select macro, then click run. Next, repeat this for Macro2 by clicking view then macros, selecting Macro2 and clicking run. In the amplification machine software, check the curves for each sample and control against the macro table by selecting analysis/amplification plot on the left side of the screen.

Then, select the sample tab and click on each sample to confirm that there are two or three positive amplification curves for each of the positive results in the table. Fluorescence readings are plotted against amplification cycles to illustrate that real-time PCR was successfully performed. Most samples reached relative threshold levels between cycles two and 25 of the real-time PCR reaction.

Standard curves run on three different days are plotted to demonstrate the linearity and range of amplification using positive controls. Cycle threshold values are plotted against log base 10 of the number of copies of nucleic acid standard. The two macros will fill in the sample names in the first column and the average of the three Ct values for each target for those with positive results.

Targets that did not amplify appear blank in this table. Quantitative results for 10 clinical samples and four routine controls are shown, 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. One sample in this set shows a failed internal control, which indicates the presence of inhibitors in the sample.

After watching this video you should have a good understanding of how to perform a nanoscale PCR experiment. This procedure will make it easier to run syndrome based panels in veterinary diagnostics. This method can be adapted to include essays for causative agents of many different infectious diseases.