A Duplex Digital PCR Assay for Simultaneous Quantification of the Enterococcus spp. and the Human Fecal-associated HF183 Marker in Waters

The overall goal of this Duplex Digital PCR Assay is to simultaneously quantify general and human associated fecal contamination in waters. This Assay can help answering key questions in recreational water quality monitioring such as, how much general fecal indicator, i.e. Enterococcus and more importantly human fecal associated HF183 Marker is in the waters.

The main advantage of this Assay is that it quantifies two target in one reaction and compared to qPCR, it eliminates the need for running standard curves and hence the associate bias and variability. Demonstrating the procedure will be Meredith Raith, our Senior Research Technician. To begin this procedure, first prepare 100 Micromole per liter stock concentrations for all primers in Molecular Grade Water and probes in TEPH 8 Buffer as described in the protocol text.

Next, prepare a Master Mix by mixing appropriate amounts of Digital PCR mix, forward and reverse primers, the fluorescent probe, and Nuclease-Free Water. Pipette up and down at least 10 times to mix while taking caution not to introduce air bubbles in the solution. Since dPCR Master Mix is more viscous than conventional qPCR Master Mix, it's necessary to use mixing by pipetting technique in order to create a homogenous solution.

This allows for accurate dPCR quantification downstream. To make the Assay Mixture for Droplet Generation for running samples in duplicate, pipette 36 microliters of the Master Mix into a regular PCR plate. To each of the 36 microliter Master Mixes, mix in 12 microliters of DNA template leaving the corresponding replicate wells empty on the plate.

Include positive controls to ensure the Assay is running properly. Include No Template Controls or NTCs to ensure there is no contamination within the plate and to set the fluorescent baseline later for data analysis. Prior to setting up the Droplet Generator mix the Assay Mixtures by using a multichannel pipette to pipette the mixtures up and down approximately 15 times.

Ensure that the pipette tip stays within the liquid to avoid making access bubbles within the mixture. Next, insert cartridge one containing eight wells into a white cartridge holder and click the cartridge holder shut. Cartridge one is now firmly in place and cannot be dislodged from the holder while generating droplets.

Using a multichannel pipette, gently transfer 20 microliters of the Assay Mixture into the middle position of the cartridge marked sample without introducing air bubbles. Pipette in 70 microliters of Droplet Generation oil to the left side of the cartridge marked oil. Cover the cartridge with a gasket making sure the gasket is flat and held evenly by the four ticks toward the edge of the cartridge.

Press the green-lit button on the Droplet Generator to open the door and place the cartridge. Press the button again to close the generator. Once the door closes, the green button is dimmed and the door cannot be reopened.

Droplet Generation will begin immediately and continue for approximately one minute. While the Droplet Generation is in progress, place cartridge two in a second white cartridge holder and prepare it in the same manner as for cartridge one. When the Droplet Generation is complete, the dimly-lit button will turn green.

Open the Droplet Generator door, remove the white cartridge holder containing cartridge one and set it aside. Place cartridge two into the Droplet Generator. Remove the gasket from cartridge one and discard.

Do not un-click the white cartridge holder as the action may break the newly generated droplets. Using a multichannel pipette set to 40 microliters, insert the tips into the third column of the cartridge marked droplets at a 45 degree angle and slowly pipette up all the droplets. Transfer them to the final PCR plate by putting the pipette tip against the well wall approximately halfway down and expelling the droplets slowly.

In the same way, when the Droplet Generation for cartridge two is complete, remove the gasket from cartridge two and transfer the generated droplets to the final PCR plate. Place a pierceable foil cover on top of the plate and place it on a plate sealer. Set the sealer to 180 degrees celsius, press play on the sealer, and seal for 10 seconds.

To begin this procedure place the sealed final PCR plate in the thermocycler. Use a thermocycler that is compatible with the final PCR plate and with a temperature ramping speed of two degrees celsius per second. Run the following thermal program ten minutes at 95 degrees celsius followed by 40 cycles of 30 seconds at 94 degrees celsius and 60 seconds at 60 degrees celsius followed by a ten minute hold at 98 degrees celsius.

Upon completion of cycling, transfer the plate to a Droplet Reader for automatic measurement of fluorescents in each droplet in each well. Ensure the droplets are at room temperature before proceeding with Droplet Reading. Start by opening the accompanying software to set up the Droplet Reading.

In the default setup menu containing a schematic of an empty 96 well plate, double click on well A1 to open the menu containing three sections, Sample, Assay 1 and Assay 2. In the Sample section, type the Sample ID into the box labeled Name and check the box to the right marked Apply. Next, click the drop down menu labeled experiment, choose RED for Rare Event Detection and click Enter.

Move to the section denoted Assay 1. In the Name section, fill out the Assay and click Enter. In the box below labeled Type, click the drop down menu, chose Channel 1 Unknown and click Enter.

Move to the section denoted Assay 2. In the Name section, fill out the Assay and click Enter. In the box below labeld Type, click the drop down menu, choose Channel 2 Unknown and click Enter.

All the information from the previous steps is now present in well A1.Name all subsequent wells containing droplets. To save total setup time, click shift or control to choose multiple wells simultaneously. When the digital depiction of the plate mirrors the physical plate, press OK at the bottom right of the menu.

In the new menu that appears at the top of plate schematic, under the Template choose Save As and name and save the plate. To the left of the screen, click Run and select the appropriate Dye Set in the pop out Run Options window. Data collection will initiate and is displayed in real time in the software.

When the reader is finished and a box appears stating Run Complete, click OK.Check the separation between the positive and negative droplets. Ensure that the fluorescents in all droplets in the NTC wells is near baseline. Click on the button labeled Events.

At the bottom, click the box named Single and to the right of the presented Histogram, click the box named Total to diplay the total number of accepted droplets per well. Exclude any wells containing less than 10, 000 droplets by holding the control key and clicking on the wells to be excluded. Click on the button denoted 1D Amplitude to set the fluorescent threshold at approximately one standard deviation of the negative droplets in the NTC wells for both targets.

At the very left of the screen under the Auto Analyze showing two threshold buttons, chose the icon to the right that has a solid pink horizontal line running through it. Click on the box to the left of Set Threshold under each of the Amplitude Graphs and enter the appropriate fluorescence threshold values. The target concentrations in copy per microliter reaction are then automatically calculated.

Export the results in a CSV file by clicking the Export button in the upper lefthand corner on the 1D Amplitude screen. In the CSV file, multiple the exported target concentration by four to convert it from copy of target per microliter reaction, to copy of target per microliter DNA template. This figure compares the duplex and simplex formats of the Digital PCR Assay.

The left and right panels display Enterococcus and HF183 Quantification respectively with the corresponding correlation coefficients between duplex and simplex results. The solid lines indicate the regression lines and the grey shading indicates the corresponding standard errors. Different types of samples are indicated by the symbols.

Results are highly consistent and often indistinguishable whether Enterococcus and HF183 are measured simultaneously in one reaction or separately in two reactions. The Digital PCR Assay can also tolerate inhibitor at concentrations one to two orders of magnitude higher than that tolerated by its qPCR counterparts. This figure shows qPCR and Digital PCR Quantification of the HF183 marker in clean sewage DNA spiked with increase in concenstrations of humic acid which inhibits PCR.

The expected HF183 Quantification in the absence of inhibitors is defined as a 95%confidence interfold between the two horizontal dotted lines. Digital PCR denoted by triangles continues to provide accurate quantification up to a humic acid concentration of 15 nanograms per microliter reaction while qPCR denoted by crosses starts to underestimate at one nanogram per microliter and becomes completely inhibited at five nanograms per microliter. After watching this video, you should have a good understanding of how to perform this enteral HF183 Duplex Droplet Digital PCR Assay or other similar Assay using different primers and probes.

Since the development of this Duplex Assay which was published in water research in 2015, we have developed and validate a slew of other Digital PCR Assays that target general fecal indicator bacteria, microbial tracking markers, and waterborne pathogens. These Assays provide direct and unbiased quantification of their target and they're becoming useful alternatives to qPCR Assays in the Water Quality Testing field.