Concentration of Virus Particles from Environmental Water and Wastewater Samples Using Skimmed Milk Flocculation and Ultrafiltration

Water-based and wastewater epidemiology has emerged as an alternative method to monitor and predict the course of outbreaks in communities. The skimmed milk flocculation method represents an alternative approach to concentrate microbes. Skimmed milk flocculation method represents a versatile, cheap, and easy hands-off method to concentrate microbial fractions with no significant differences compared to ultra filtration or tangential flow filtration approaches.

This approach is used for metagenomics or targeted PCR studies. Once the microbial fractions are concentrated, nucleic acid extraction can be conducted using commercial extraction kits or in-house protocol. The skimmed milk flocculation method is relatively easier for large or small volumes of water or wastewater samples.

A background control with Milli-Q water should be included as part of this method. Demonstrating the procedure will be Kadir Yanac, Jhannelle Francis, and Jocelyn Zambrano-Alvarado, graduate students from Miguel Uyaguari's laboratory. To begin, collect two liters of 24-hour flow proportional composite raw wastewater samples.

Transport the samples to the laboratory in light-proof bottles in an icebox and process them within 24 hours. Get the wastewater characteristics data from wastewater treatment plant technical staff. For each ultra filtration method, spike five times 10 to the fourth copies of armored RNA into 140 milliliters of each of six fresh wastewater samples collected on one date and six fresh wastewater samples on another date.

Homogenize the armored RNA in the sample by stirring for 30 minutes at four degrees Celsius on a magnetic stir. For skimmed milk flocculation or SMF, prepare 1%skimmed milk solution by dissolving 0.5 grams of skimmed milk powder in 50 milliliters of synthetic seawater. Adjust the pH of SMF to 3.5 using one normal hydrochloric acid.

Add five milliliters of skimmed milk solution to 500 milliliters of raw wastewater sample. Stir the sample for eight hours and allow the formed flocks to settle for another eight hours at room temperature. Remove the supernatant using a serological pipette without disturbing the settled flocks.

Transfer a final volume of 50 milliliters containing the flocks to a centrifuge tube and centrifuge at 8, 000 G for 30 minutes at four degrees Celsius. Remove the supernatant and carefully scrap the pellet using a sterilized spatula. Resuspend the remaining pellet in 250 microliters of 0.2 molar sodium phosphate buffer.

Transfer the scraped and resuspended pellets to the same 1.5 milliliter mini centrifuge tube. Perform viral RNA extraction from viral concentrates of wastewater samples and recovery efficiency assays using an RNA extraction kit with a 25:24:2:1 ratio of phenol to chloroform to isoamyl alcohol and beta mercaptoethanol according to the manufacturer's instructions. Finally, elute the RNA in 50 microliters of elution buffer.

For RT-qPCR analysis, quantify armored RNA using tenfold dilutions of a synthetic single stranded DNA or gBlock construct. Obtain calibration curves for each RT-qPCR run. Include negative controls and run the samples in triplicate.

Collect 10 liters of environmental surface water sample. Include 10 liters of deionized water for background control. Perform capsule and vacuum filtration using membrane disc filters of 0.45, 0.2, and 0.1 micron sizes to minimize noise during metagenomics sequencing.

After filtration, adjust the pH of the water sample to 3.5 using one normal hydrochloric acid. Prepare a 1%pre-flocculated skimmed milk solution and adjust the pH to 3.5. Add 100 milliliters of the acidified skimmed milk solution to 10 liters of acidified environmental water sample.

Stir the sample for eight hours at room temperature on a magnetic shaker and allow the flocks to sediment by gravity for an additional eight hours. After settling, carefully remove the supernatant using a vacuum pump without disturbing the flocks. Aliquot and balance the remaining flocks according to the weight in a 50 milliliter centrifuge tube and centrifuge.

Remove the supernatant and dissolve the pellet in 200 microliters of 0.2 molar sodium phosphate buffer. Treat the dissolved pellets with DNase I and RNase A following the manufacturer's instructions to eliminate free DNA and RNA coprecipitated during the extraction process. After incubation, inactivate DNase I and RNase A.Extract nucleic acids from the dissolved pellet using a DNA or RNA extraction kit.

Quantify the total amount of extracted DNA or RNA from the environmental surface water sample using a fluorometer. Perform qPCR analysis to target enteric viruses of interest and Illumina sequencing to identify the viral community structure. Collect two liters of water samples from protected and impacted waterways.

If the sample contains considerable debris, use a cheesecloth to remove them. Add 20 milliliters of 1%skimmed milk solution to the previously pH adjusted two liter freshwater sample. Stir the sample for eight hours at room temperature and allow the flocks to sediment by gravity for an additional eight hours.

After settling, carefully remove the supernatant using a peristaltic pump. Transfer the sedimented flocks to a 50 milliliter centrifuge tube and centrifuge them at 8, 000 G for 30 minutes at four degrees Celsius. Discard the supernatant and resuspend the pellets in 200 microliters of 0.2 molar sodium phosphate buffer.

Select around 0.5 grams of the flock for DNA extraction using the nucleic acid isolation kit of choice following the manufacturer's instructions. Determine the DNA concentration and purity using a fluorometer. All six samples processed with ultra filtration at 3, 000 G were positive.

In contrast, only one sample was positive when the samples were processed with ultra filtration at 7, 500 G.All samples processed with SMF were positive and resulted in better recovery. The average recovery rates of 3, 000 G and SMF were significantly and consistently higher than ultra filtration at 7, 500 G.Water quality parameters in influenced samples and nucleic acids extracted from environmental water samples collected within Winnipeg across three seasons are summarized here. Samples collected from April to October 2022 from Assiniboine River water yielded the highest DNA concentration from Forested 1 and the lowest concentration was recovered from agricultural site three.

It is very important to use the right concentration of skimmed milk and acidify the sample prior to use. This technique can provide a quick screening of different microbial pathogens present in environmental samples such as water and wastewater. Additional modifications can be implemented using solid matrices such as soil or sediments.