The overall goal of this procedure is to measure the enterovirus and norovirus concentration in water, using reverse transcription quantitative polymerase chain reaction. This is a accomplished by tertiary concentration of virus particles from water samples concentrates using centrifugal ultrafiltration. Then, standard curve reagents are prepared as described in the protocol.

Followed by nucleic acid extraction of viral concentrates and standard curve. Then the viral RNA and one to five and one to twenty five-fold dilutions of the RNA are reverse transcribed using random primers and reverse transcriptase. Finally, amplification of viral cDNA is carried out using virus specific primers and probes in a real-time thermal cycler, followed by sample genomic copy calculations based on the standard curve.

The main advantage of EPA method 1615 over other standardized methods like ISO 15216 is that it's designed specifically for water samples and includes an enterovirus assay. This method can provide risk assessors in the environmental microbiology field with the current data for determining risk from viruses and drinking in recreational waters. Generally, individuals new to this method will struggle with the standardization built into the method.

The method is very detailed and must be followed explicitly. Visual demonstration of the method is critical because even slight variations in the method can cause negative consequences. Demonstrating the procedure today are Nichole Brinkman, Jennifer Cashdollar, Shannon Griffin, and myself, Shay Fout.

We're all professional staff members of EPA's national exposure laboratory in Cincinnati, Ohio. After preparing a standard curve according text protocol, prepare a centrifugal concentrator for each sample collected by adding at least 10 milliliters of 1X PBS and 2%BSA to the upper sample chamber. After incubating overnight, washing, then adding the samples as outlined in the text protocol, centrifuge each test sample in a swinging bucket rotor at 3000 6000 times g and four degrees Celsius for twenty to thirty minutes.

Once the sample has been appropriately concentrated and transferred to a fresh microcentrifuge tube, add 15 molar sodium phosphate buffer to adjust the final volume to 400 microliters plus or minus two microliters. To extract the nucleic acids, add 200 microliters of extraction buffer for a negative extraction control and 200 microliters of the tertiary concentrate for each test sample or standard curve dilution to separate microcentrifuge tubes and extract the RNA according to the modifications in the text protocol of the extraction kit manufacturer's instructions. Following the preparation of primers and probes as outlined in the protocol, prepare reverse transcription master mixes one and two in a clean room, as shown here.

Using a multichannel pipette, transfer 16.5 microliters of RT master mix one into the wells of each PCR plate. Next, with the nucleic acid extration kit's elution buffer containing 400 units per mililiter of RNAse inhibitor, dilute the thawed field and lab fortified sample matrix or LSFM samples one to five and one to twenty five. Transfer 6.7 microliters of the nucleic acids from every test sample, control, and standard curve into separate PCR plate wells using triplicate wells for test samples and controls and duplicate wells for standard curves.

Add 6.7 microliters of elution buffer into no template control or NTC wells. Include two to eight NTCs per RT plate, using two for the first sample and then two or more for every fourth additional sample, distributing the negative controls throughout the plate. Use the heat resistant plate sealer to seal the PCR plate, making certain that each well is tightly sealed.

Then, mix the samples for five to ten seconds before briefly centrifuging at greater than or equal to 500 times g. In a thermocycler, incubate the plate at 99 degree Celsius for four minutes. Bring the plate to four degrees Celsius and then briefly centrifuge a second time.

Then, carefully remove the plate seal and add 16.8 microliters of RT master mix two to each well. After sealing the plate and briefly mixing and spinning, place the plate in the thermocycler and run at 25 degrees Celsius for 15 minutes followed by 42 degrees Celsius for 60 minutes, 99 degrees Celsius at five minutes, and finally, hold at 4 degrees Celsius for up to eight hours or store samples at 70 degrees Celsius for a longer period. In a clean room, prepare quantitative PCR master mixes using the guides for enterovirus, norovirus genogroup one, novovirus genogroup two, and hepatitis G.Mix each master mix and briefly centrifuge.

Then, as shown in this example, add 14 microliters of the PCR master mixes to the appropriate wells of a labeled optical reaction plate using separate plates for each qPCR assay. Dispense six microliters of the appropriate cDNA to the appropriate wells. Then mix the samples and briefly centrifuge.

Set up the thermocycler software as described in the protocol and run the hepatitis G qPCR assay on the undiluted and diluted field and lab fortified sample matrix or LFSM samples before running all other qPCR assays. Compare the mean Cq value of each sample to the mean hepatitis G Cq value predetermined for each new lot of hepatitis G reagent. Use the lowest dilution of field or LDF sample that is less than one Cq value greater than the mean hepatitis G Cq value for the enterovirus and norovirus qPCR assays.

Set up the quantitative PCR thermocycler software according to the manufacturer's instructions. Identify the standard curve samples as standards and for each standard curve dilution, enter the genomic copy values shown here. Run the plate at 95 degrees Celsius for ten minutes followed by 45 cycles of 90 degree Celsius for 15 seconds and 60 degrees Celsius for one minute.

Determine whether each standard curve meets the acceptable values given in this table. After calculating standard deviation, slope, R squared, and percent efficiency according to the equations in the text protocol, record the genomic copy or GC values calculated by the thermocycler software for all the test samples based upon standard curves that meet the criteria specified in this table and the mean GC values for each sample. Determine the GC per liter for each test sample using the following equation where GC is the mean genomic copy number just calculated.

The factor 199 is the total dilution factor for the volume reactions that occurred during the tertiary concentrations. RNA extraction and RT-qPCR are steps. DF is the dilution factor that compensates for inhibition and D is the volume of the original water sample assay in liters.

Finally, compute the total GC of lab field fortified blank or LFB and lab reagent blank or LRB samples by multiplying the mean GC value previously determined by 199 and dividing by 3. Overall, virus recovery was determined using paired field LFSM ground water samples from a total of seven sample sets recovered from three public treatment plans and a private well. Seed levels for the LSFM samples were three million MPN of seven poliovirus type three and five million PFU of murine norovirus.

Murine norovirus was used for the initial single laboratory evaluation of EPA method 1615 because the standard curve reagent was not available at the time. For ground water samples, the mean poliovirus recovery was 20%with a standard error of 2%while mean murine norovirus recovery was 30%with a standard error of 3%The regular field groundwater sample for each LFSM had no detectable enterovirus or norovirus. Lab fortified blank or LFB and lab reagent blank or LRB samples were measured using seeded and unseeded reagent grade water.

Poliovirus recovery averaged 44%with a standard error of 1%while murine norovirus recovery averaged 4%with a standard error of 5%This figure shows typical standard curves for enterovirus and norovirus genogroup two. The novovirus genogroup two curve meets the standard curve criteria with an R squared value of 9987, overall standard deviation of 14 and 101%efficiency. The enterovirus curve meets the standard curve performance criteria with an R squared value of 9874 an overall standard deviation of 58, and 103%efficiency, but has about 100-fold less sensitivity and thus a higher detection limit than the norovirus curves.

After watching this video, you should have a good understanding of how to concentrate and detect waterborne enterovirus and norovirus using RT-qPCR. Once mastered, this technique can be done in about eight hours for low turbidity samples. For samples with higher turbidity, they may require additional time, especially at the tertiary concentration step.

While performing this procedure, it is important to protect samples from RNAse contamination prior to reverse transcription. Don't forget that PCR is a very powerful and useful technique. Precautions such as separation workstations and use of proper controls is necessary in order to minimize false positive and false negative results.