EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. I. Collection of Virus Samples

The overall goal of this procedure is to provide a standardized approach for sampling environmental and drinking waters for occurrence of enteric viruses based on EPA method 1615. This is accomplished by first assembling a sampling apparatus, sterilizing the intake and cartridge housing modules of the apparatus, and then aseptically adding a sterile electropositive cartridge filter. Next, a defined volume of water is passed through an electropositive filter contained in the sampling apparatus.

Then the excess water is drained from the apparatus and cartridge filter housing, and the volume sampled is recorded on a sample data sheet. Finally, the sampling apparatus components are packed in a cooler shipping container kept at one to 10 degrees Celsius and delivered to an analytical laboratory. Ultimately, enteroviruses and noroviruses in environmental and drinking waters are measured using this sampling procedure as part of an overall method.

This procedure uses Electropositive filter to concentrate viruses, some environmental drinking waters. There are several other filter types that can be used to do this procedure. One is the ultra filter and another is a negatively charged filter.

The vantage of the electro Posy filter over the ultra filter is that the sampling is about two and a half times faster. Virus collection using negatively charged filters requires pH adjustment in the addition of salts. The advantage to positively charge filters is that no adjustments are needed prior to sample collection.

Generally, individuals new to this procedure will struggle with maintaining sterility and with unexpected conditions, such as the absence of a convenient tap for sample collection. Demonstrating the procedure today will be myself, sha out, Jennifer Cash dollar and Eunice VER geese. We are all professional staff members of EPAs National Exposure Research Laboratory in Cincinnati, Ohio.

To prepare for sample collection, assemble a standard filter apparatus as shown here consisting of an intake module, a cartridge housing module, and a discharge module as described in supplemental materials of the text protocol. Using the flow rates for sampling. Calibrate the flow meter totalizer before the first use by connecting a discharge module to an intake module, and then connecting the intake module to a tap.

Turn on the tap completely and then use the bronze globe valve to adjust the flow rate to 10 liters per minute. Once the flow rate is stable at 10 liters per minute, reset the flow meter totalizer to read total volume. Next place the outlet of the discharge module into a four liter or larger graduated cylinder, and simultaneously reset the totalizer to zero.

Measure the totalizer reading at the four liter mark and the timer required to reach the four liter mark on the cylinder. If the totalizer reading is less than 3.96 or more than 4.04 liters, calculate a correction factor needed to adjust for the observed difference. For example, if the flow meter totalizer reads 3.9 liters at the four liter mark on the graduated cylinder, the correction factor would be 1.026 or four divided by 3.9 To sterilize the standard filter apparatus and prepare it for sample collection, use 0.525%sodium hypochlorite to completely immerse the intake and cartridge housing modules and soak for at least 30 minutes.

Alternatively, use a pump to circulate the solution through the modules. Use sterile DH two O to rinse the modules, and then use a sterile solution of 50 milliliters of one molar sodium thio sulfate per liter of sterile reagent grade water to de chlorinate for five minutes. Then use sterile aluminum foil to cover the filter sampling apparatus.

Module ends aseptically. Add an electropositive cartridge filter to the cartridge housing module taking care that it is properly seated. In the housings record a unique sample number on a sample data sheet, and then take or ship the filter sampling apparatus, the sample data sheet, and any necessary instructions concerning sample collection to the individual who will be collecting the field sample Upon arrival at the collection site and after washing hands, dawn surgical gloves to minimize sample contamination.

Change gloves frequently, especially after touching water taps and other items that may be contaminated. Use a pad containing sodium hypochlorite to sterilize the tap to the sampling apparatus and inner threads on a garden hose. Record relevant sample tracking information on a sample data sheet, including site and sample identification, sampler name, equipment models, and serial numbers.

Attach the garden hose to the tap. Place the outlet of the hose in a place where the water can be safely drained, and then turn on the water. Tap for two to three minutes to clear any debris that has settled in the line.

Connect the discharge module to the intake module, and then connect both to the water tap. Connect a garden hose to the end of the discharge module and place the free end in the one liter propylene container. Next turn on the tap and pass at least 75 liters of the water to be sampled through the apparatus.

Measure and record the following water quality parameters during the flush period on water flowing into the propylene container, chlorine residual pH, temperature, and turbidity. After turning off the water at the tap, disconnect the discharge module from the intake module and then connect the cartridge housing module to the outlet of the intake module and the discharge module to the outlet of the cartridge housing module. Then reset the totalizer reading to zero.

To begin field sample collection. Record the initial totalizer reading along with the date and time, and then slowly open the water tap. Ensure that the cartridge housing is in an upright position and that it completely fills with water.

Now, open the tap completely and use the bronze globe valve to adjust the flow rate to 10 liters per minute Before recording the initial flow rate. Using the totalizer readings, pass 300 liters of surface water or 1500 to 1800 liters of groundwater through the apparatus. Observe the flow rate as the sample volume approaches the end of the sampling period, and then turn off the flow of water at the sample tap record the final flow rate, date, time of day, the final totalizer reading and the total sample volume.

Disconnect the filter sampling apparatus from the tap. Disconnect the cartridge housing module from the other modules. Turn the filter housings upside down and allow excess water to flow out until no more water will drain from both the inlet and outlet ports.

Turn the housing upright and use sterile foil to cover the quick connects on each end. Place the housing into one or more sealable plastic bags. Drain the water from the intake and discharge modules.

Place the modules into one or more sealable plastic bags to prepare the samples for shipping. Place the filter sampling apparatus modules into an insulated storage and transport cooler. Add frozen ice packs or double bagged ice cubes to the storage and transport cooler to ensure that the sample remains between one to 10 degrees Celsius.

During shipment. Fill any void space with packing material and then place the sample data sheet in a sealable plastic bag and place it on top of the packing material. Close the insulated storage and transport chest transport, or ship the sample to the analytical laboratory.

If the chest is to be shipped, seal it with tape to prevent leakage. Ensure that the sample arrives at the analytical laboratory no later than 24 hours after the start of field collection. Filter clogging is a major potential problem that can be encountered with Method 1615 since it decreases the flow rate of water.

This table shows the percentage of samples with reduced volumes as a function of filter type, water type, and turbidity. When filtering groundwater samples, the filter containing nano Illumina fibers outperformed a quaternary amine based filter. The ary amine based filter was better for filtering surface water samples.

Overall, 6%of samples collected using the quaternary mean based filter were deficient in volume. While 10%of samples collected with the aluminum oxide, nanofiber based filters failed to meet the required minimum specified volume due to clogging. While attempting this procedure, it’s important to remember to maintain sterile conditions.

This is just the first procedure of an overall method. Following this procedure will be filter elution, secondary concentration, and assays such as the total cultural virus, assay, and molecular assays. These later assays are needed to actually measure virus occurrence and concentration.

After watching this video, you should have a good understanding of how to collect virus samples from environmental and drinking waters. Don’t forget that working with viral pathogens can be hazardous, and you should always take precautions such as the use of gloves while performing this procedure.