Kosteneffectieve methode voor Microbiële Source Tracking Met behulp van specifieke menselijke en dierlijke virussen

Cost effective method for microbial source tracking using specific human and animal viruses. The study describes a cost effective method for the identification of the source of fecal urine contamination or contamination by nitrates in water using quantitative PCR for the specific quantification of human porcine, bovine, DNA viruses, adenoviruses, and polyomaviruses proposed as microbial source tracking tools. We have developed a protocol for the concentration of viruses from 10 liter water samples by organic flocculation using skimmed milk extraction of viral nucleic acids from the sediment and quantification of human bovine or porcine DNI viruses using quantitative PCR.

It is well known that microbial contamination of the vitamin represents a significant health risk and we need to know the sources of the contamination. We have proposed the use of highly prevalent DNA viruses as microbial source tracking tools. The selected viruses are adenoviruses and polyomaviruses.

Those viruses are being excreted persistently all over the year and in all geographical areas. Studied in previous studies. We have developed robust low cost concentration methods for viruses in water, and we have been working developing QPCR assays for the quantification of purine adenoviruses and bovine polyomaviruses.

In addition, we use human adenoviruses, NGC Polyomaviruses, tested by QPCR, also as markers of human contamination in water, Collection of water samples. In this study, four replicates of 10 liter per water sample are collected in plastic containers with flat bottom and one extra sample as a process control. This last sample will be seeded with a known amount of the viral particles used as a control.

A negative control is prepared in the laboratory by using condition tap water in one extra plastic 10 liter container. If the sample presents high quantity of suspended material, sand and other materials, let it sediment for 15 minutes, transfer the water into a new container. Viral concentration by organic flocculation.

Detection of viruses in low or moderately polluted water samples requires the concentration of the viruses from at least several liters of water into a much smaller volume. The concentration procedure includes acidification of 10 liter water samples flocculation using skimmed milk gravity, sedimentation of the flocks and collection of the precipitate in 10 milliliters of phosphate buffer to start the process, a solution of locating skim milk in artificial sea water is prepared by dissolving 10 grams of skim milk powder in one liter of artificial sea water and carefully adjusting the pH to 3.5. With hydrochloric acid, the flock should be visible.

Prepare the solution just before to be used or store at four degrees Celsius for 24 hours. The water samples are stirred and the conductivity in the samples is measured. If it is below 1.5 millisiemens, sea salts will be added.

Adjust the pH of the water sample to 3.5 by the addition of hydrochloric acid. Record the pH of the samples before and after conditioning, as well as the volume of hydrochloric acid used. The conductivity also should be recorded.

After adjusting the pH, we add 100 milliliters of pref flod skim milk, 1%to the 10 liter water sample, and stir the samples for eight to 10 hours to allow the viruses to absorb to the flocks. For the spike sample used as process control, add the standard volume of the control virus, one milliliter, for example, to the sample. Stop the stirring and let the flock sediment by gravity for eight to 10 hours.

After 16 hours, commonly the following day, we will be able to collect the sediments. To do so, remove the supernatant using a peristaltic pump. Collect the sediment with the flocks, approximately 500 milliliters into a centrifuge bottle.

Balance the pots by the addition of pref flod skim milk pH 3.5, centrifuge the pots with high speed centrifuge, 8, 000 Gs 30 minutes at four degrees Celsius. As soon as the centrifuge stops, remove the centrifuge pots carefully from the centrifuge. Very gently pour off and discard the supernatant.

Dissolve the pellet in each centrifuge bottle to a final volume of 10 milliliter phosphate, nucleic acid extraction and quantification of viruses. The viral concentrate is used for the extraction of viral nucleic acids and the specific adenoviruses and polyoma Viruses of interest will be quantified by A-Q-P-C-R. Perform a nucleic acid extraction with the key amp viral RNA mini kit.

The lysis of the viruses present in 140. Microliters of viral concentrates is performed manually, and total nucleic acids are extracted to 80 microliters. This kit enables the use of an automated platform such as Key cube.

The next step is the quantification of viral genome. Copies in the samples by using a quantitative PCR with tac man probes, human adenoviruses, JC Polyoma viruses and bovine polyoma viruses may be quantified in the same 96 well plate. Since all use the same cycles of amplification, porcine adenoviruses should be tested separately in an independent plate for the quantification of the target virus.

Prepare the quantitative PCR mix in a clean separate area. Once the mix has been prepared, allocate 15 microliters into each well, including the controls. Add the nucleic acid extractions from the samples 10 microliters in a separate area, run direct and a tenfold dilution in purified water of each sample in duplicate the total volume for one reaction after addition of target will be 25 microliters, 15 of mix plus 10 of sample or standard cover the wells containing the samples with part of an adhesive cover.

Keep the other part of the cover for the following step. AB dilution of the DNA standard suspensions. 10 microliters from 10 to zero to 10 to six genome copies, pretend microliters by triplicate and using a micro pbit exclusively used for the standard DNA cover.

The wells containing the standards with the adhesive cover previously cut. This figure shows the distribution of the standard suspension samples and controls. In the 96 well plate.

Perform the QPCR into an adequate system, selecting the appropriate parameters following activation of the amplitude gold for 10 minutes. At 95 degrees Celsius, 40 cycles of amplification are performed once the reactions are completed. Store data and results as described in the user's manual of the equipment used.

The amount of DNA will be defined as the median of the data obtained after correcting the dilution factor when needed. The amplification plot and standard curve obtained for the quantification of porcine adenoviruses showed a correlation coefficient of 0.999. Acceptable slope values range from minus 3.1 and minus 3.6 following the procedure.

Described human and animal viruses have been tested in groundwater samples from an area presenting high levels of nitrates to define the sources of contamination. The four replicates analyzed showed positive results for porcine adenoviruses mean value, 7.74 per 10 to two genome copies per liter, which would be related to the presence of porcine slurries in the area surrounding the sampling site, and would prove the presence of fecal porcine contamination in groundwater. No human contamination was present in any of the tested samples.

The virological data was confirmed by nested PCR and sequencing analysis.Conclusion. In this study, we have presented results analyzing groundwater samples, presenting contamination of porcine origin in absence of human or bovine contamination. The procedure has also been applied to the analysis of bathing waters, sea water, and river water.

We present here the procedure for applying these tools to the identification of the contamination source in groundwater, presenting high levels of nitrate as an example of the applicability of the developed methods to detect the source of contamination in the environment.