Grantee Research Project Results
Final Report: Rapid Concentration of Viruses from Water
EPA Contract Number: EPD10063Title: Rapid Concentration of Viruses from Water
Investigators: Wong, Choi-lok Rebecca
Small Business: Scientific Methods, Inc.
EPA Contact: Richards, April
Phase: II
Project Period: May 1, 2010 through April 30, 2012
Project Amount: $224,987
RFA: Small Business Innovation Research (SBIR) - Phase II (2010) RFA Text | Recipients Lists
Research Category: Small Business Innovation Research (SBIR) , SBIR - Water and Wastewater
Description:
The basic platform of two water sampling and concentration devices (one column based, and the other based on canister design), was modified to allow for 10-40 L of water to be sampled. During this period, viruses were attracted to anionic resins that were used to concentrate the viruses from the water. The amount of resin used to concentrate the viruses was optimized, which allowed for effective downstream isolation of the target nucleic acid. One of the water sampling and concentration devices was further modified such that a resin capture filter was included which functioned to capture the resin and any attached viruses following water sampling. Nucleic acid was isolated directly off the resin within the filter in an efficient manner. A rapid, field based syringe method for total nucleic acid isolation was developed which allowed for isolation from the resin filter to be achieved within 20 minutes. Real time PCR assays and loop mediated amplification assays were developed for detection of the viruses using the isolated nucleic acid as a template. In some cases, detection was achieved within 15 minutes. The integrated sampling, concentration, and detection methods could lead to a result from start to finish within 4 hours, and was tested as described below.
Several enteric viruses were seeded into water at different concentrations, followed by analysis of the ability of the resin to concentrate the viruses from the water. The viruses used in this study included Coxsackie virus, feline calicivirus and Adenoviruses 40 and 41. All virus capture experiments were conducted in a batch format or semi-batch format, with 0.5 g of anionic exchange resin beads added to 10 liters (batch) or 40 liters (semi-batch) of tap or sea water that contained various concentrations of the viruses. Following viral capture and concentration, the syringe based nucleic acid isolation method was developed that enabled the nucleic acid to be isolated from viruses directly bound to the resin beads, which were contained within the resin filter, without the need to elute the viruses from the beads. This isolation method is unique, because it reduces the presence of inhibiters (often found in elution buffers) that affect downstream molecular based (PCR) assays, ultimately increasing the sensitivity of the assay. Following the concentration and nucleic acid isolation steps, FRNA bacteriophages (as models of enteric viruses) were detected by RT-PCR and LAMP. The enteric viruses were detected by RT-PCR. Field based detection of the FRNA bacteriophages was demonstrated in South Africa, Mexico, and the Gulf of Mexico at several oyster growing beds.
Summary/Accomplishments (Outputs/Outcomes):
The redesigned concentration devices allowed the anionic exchange resin to efficiently concentrate all viruses from the tap water and sea water in volumes ranging from 10-40 L of water, regardless of whether the format (batch or semi-batch, or column) used to concentrate the viruses. The efficiency of capture was greater than 90% for all viruses. When used in conjunction with the anionic exchange resin beads and RT-PCR or LAMP, the concentration devices enabled rapid detection of viruses from the water within 4-8 hours. The syringe based nucleic acid isolation method worked well and permitted nucleic acid isolation from all viruses. The RT-PCR method also worked well and allowed for detection of as low as 10-2 PFU/ml from water samples. The RT-PCR assays could run simultaneously in individual wells of the same 96 well PCR plate. By the end of this research project, a simple system for virus concentration has been constructed.
Capture efficiency of MS2 (model RNA virus) and ΦX174 (model DNA virus) in 10 liter ground by the automated filtration system at 300mL/min flow rate and 1” column packed with 20 g of positively charged resin beads were 99.4% and 86.2%, respectively. When sample volume was increased to 50L, the average recovery was decrease to 37%. A universal lysis buffer was developed to simultaneously extract viral DNA and RNA directly from resin beads. The detection of the single stranded DNA of φX174 was done by real time quantitative PCR using a hydrolysis probe and the detection of the MS2 RNA was done by real time RT-PCR using SYBR Green I and melting curve analysis. The results indicate that viral DNA and RNA can be simultaneously extracted from both resin and silica beads by this universal lysis buffer. The universal lysis buffer/IBI extraction kit and the IBI lysis buffer/IBI extraction kit gave the best outcome for extracting and concentrating both DNA and RNA viruses.
Conclusions:
The results indicate that the resin efficiently concentrated all viruses from the reagent, drinking, and ground waters, regardless of the water sample volume was small (500 mL) or large (10, 20 liters). The efficiency of capture was greater than 70% for all viruses. The viral RNA/DNA was then extracted directly from the resin using a flow through nucleic acid isolation method. Nucleic acid isolation from all viruses was directly used as templates in the RT-RT-PCR. All phages and viruses were detected successfully by the resin capture/nucleic acid extraction/PCR method, at sensitivities ranging from 100 to 101 PFU/ml. The detection method developed has eliminated the need for the elution and secondary concentration steps, reducing the total assay time and increasing detection sensitivity.
The study showed that the viral mix (model DNA and RNA viruses) could be captured very efficiently in 10 L of ground water by the positively charged resin beads using automatic filtration system. Viral DNA and RNA can be extracted simultaneously and detected by real-time (RT)-PCR.
Supplemental Keywords:
anionic resin beads, virus concentration, viral nucleic acid extraction, filtration, drinking water, groundwater, positively charged filter mediaSBIR Phase I:
Rapid Concentration of Viruses from WaterThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.