Grantee Research Project Results

2009 Progress Report: A Novel Molecular-Based Approach for Broad Detection of Viable Pathogens in Drinking Water

EPA Grant Number: R833011
Title: A Novel Molecular-Based Approach for Broad Detection of Viable Pathogens in Drinking Water
Investigators: Meschke, John Scott , Cangelosi, Gerard A.
Institution: University of Washington , Seattle Biomedical Research Institute
EPA Project Officer: Aja, Hayley
Project Period: July 3, 2006 through July 2, 2009 (Extended to August 31, 2010)
Project Period Covered by this Report: July 3, 2008 through July 2,2009
Project Amount: $597,987
RFA: Development and Evaluation of Innovative Approaches for the Quantitative Assessment of Pathogens in Drinking Water (2005) RFA Text | Recipients Lists
Research Category: Drinking Water , Water

Objective:

The objective of this research is to develop and evaluate a novel, molecular-based approach for broad detection and enumeration of viable pathogens in drinking water.

Progress Summary:

In this year of the project our work focused on evaluation of encapsulated, positively charge filters for concentration of viruses from water, development of an ICC-qRT-PCR methods for detection of viable +ssRNA viruses, and continued development and evaluation of microfluidic cards for nucleic acid extraction.

Work Progress:

Filter Challenge Studies:

Human enteric viruses are diffusely distributed in environmental waters, often necessitating concentration of tens to hundreds of liters for effective detection. We evaluated ViroCap disposable capsule filters (Scientific Methods, Inc., Granger, IN) for concentration of several viruses from a variety of water types. The efficiencies of the ViroCap capsule filter were compared to those of the 1MDS cartridge and the OptiCap capsule filters to determine relative performance of each filter under standard conditions. Preliminary experiments using natural waters seeded with virus were also performed to evaluate whether experimental recoveries were consistent with those from environmental matrices.

Viruses used in this study include coliphage MS2, adenovirus type 2, and poliovirus type 1. Inocula, seeded water, and eluate concentrations of MS2 were enumerated using the double agar layer (DAL) method on host E. coli F_amp. Filtrate concentrations of MS2 were enumerated using the two-step enrichment method adapted to an MPN format (USEPA method 1601). Inocula, seeded water, and eluate concentrations of PV1 were enumerated by plaque assay on BGMK cells in 60 mm dishes. Adenovirus type 2 was assayed by TCID 50 assay on A549 cells.

Three different filter media (NanoCeram^Ò, 1MDS, and OptiCap) were used in this study. NanoCeram filter media (Argonide, Sanford, FL, USA) was used in all water matrices. For this study the cartridge filter was incorporated into a 12.7 cm disposable capsule, marketed as the ViroCap (Scientific Methods, Granger, IL, USA). The other filters used as benchmarks were the 25.4 cm 1MDS pleated cartridge filter (Cuno, Inc.; Meridian, CT, USA) with a nominal 10 μm pore size, and the 10.2 cm OptiCap capsule filter containing a mixed nitrocellulose ester filter material with a pore size of 0.5 μm (Millipore, Billerica, MA, USA).

Recovery efficiencies were initially determined for DI water and artificial seawater (prepared by adjusting DI water to a specific gravity consistent with marine coastal waters (1.022-1.029) using Instant Ocean (Spectrum Brands, Atlanta, GA, USA) marine salts). Later experiments investigated recoveries from natural groundwater, surface water, and seawater (s.g. 1.022) samples.

For seeded recovery experiments, twenty liter volumes of water were inoculated with 10⁶ PFU of MS2, adenovirus, or MS2 and PV1. Prior to inoculation, viruses were dispersed by agitation, for 15 minutes in a 30 mL aliquot of the challenge water, and subsequent filtration through a 0.22 μm low protein binding syringe filter. After mixing, an aliquot of the seeded water was removed for enumeration and the remainder passed through filters using the setup depicted in Figure 1.

Fig. 1 Schematic of filtration experimental setup

Viruses adsorbed to positively charged filters were eluted with 1.5% beef extract containing 0.05M glycine with 0.01% vol./vol. Tween 80, adjusted to pH 9.5 or OptimaRE. Later experiments with adenovirus incorporated sodium polyphosphates in the eluant for improved recovery. OptiCap filters were rinsed with 500 mL of 0.5 mM H₂SO₄ (pH 3.0) and eluted with 350 mL of 1 mM NaOH (pH 10.8) and neutralized with 3 mL of 50 mM H₂SO₄ and 100 μL of 100x Tris-EDTA buffer (pH 8.0) according to a protocol adapted from Katayama, et al. (2002). Percent recovery from filters was determined by comparison of viral titer of the influent to the viral titer of the eluate.

The mean adsorption for MS2 by the ViroCap was 88%. Recovery of MS2 was significantly greater (p≤0.01) than alternative filters tested: 65% from DI water and 63% artificial seawater, compared to 30% for the 1MDS and 15% for the OptiCap for the respective matrices. Recovery of PV1 from DI water (37%) was similar to that of the 1MDS (51%). PV1 recoveries from artificial seawater were significantly greater (p≤0.01) for the ViroCap (44%) than the OptiCap (11%).

Table 1. Percent recovery of MS2

Water Type	Filter	N*	Mean ( ± St. Dev)
DI Water	ViroCap	23	65	( ± 23)
	Virosorb 1MDS	3	30	( ± 10)
Artificial Seawater	ViroCap	16	63	( ± 13)
	OptiCapXL	3	15	( ± 4.5)

* = number of trials

Table 2. Percent recovery of PV1

Virus Type	Filter	N*	Mean ( ± St. Dev)
DI Water	ViroCap	6	37	( ± 12)
	Virosorb 1MDS	3	51	( ± 5.8)
Artificial Seawater	ViroCap	6	44	( ± 25)
	OptiCap XL	3	11	( ± 2.8)

* = number of trials

Recovery of MS2 from seeded environmental samples yielded 44% from groundwater, 53% from surface water, and 51% from seawater.

Table 3. Percent recovery of MS2 from natural waters

Matrix	N*	Mean ( ± St. Dev)
Groundwater	3	44	( ± 6.6)
Surface water	3	53	( ± 4.1)
Seawater	3	51	( ± 0.3)

The novel ViroCap capsule filter is a promising, low-cost, alternative to current cartridge and capsule filters. Recoveries of coliphage MS2 from deionized water using the ViroCap filter were significantly greater than recoveries using the standard 1MDS filter, and poliovirus recoveries were comparable. The ViroCap filter was uninhibited by natural matrices and showed consistent recoveries from groundwater, surface water, and seawater.

On a related project involving an eight month investigation into the quality of water in a shallow aquifer in rural Cambodia, we field evaluated 47 mm flat disc versions of the ViroCap filters for analysis of male specific coliphage. In July of 2008, 17 active wells (eight open (O) and nine rope-pump (RP)) were identified in Kbal Kaoh and Preaek Aeng in the Kandal Province in Cambodia and selected for further study. Sampling began in late July and continued through February 2009. Samples were analyzed for Total Coliforms (TC) and E. coli by a modified membrane filtration technique on Rapid E. coli 2 Agar (BioRad, Hercules, CA, USA) for 24 hrs at 37°C. For male-specific coliphage (MsC) 1L samples were filtered serially through a Whatman 47mm filter (pore size 20-25µm) to remove particulates and then a ViroCap 47mm positive-charge filter (Scientific Methods, Granger, IN, USA). Filters were eluted with 5mL of OptimaRE buffer (Scientific Methods) with 0.01% Tween 80 and enumerated using the double-agar layer on host E. coli F_amp.

Figure 2: Microbial Quality. Contaminant level by well type and season. Shown are median, interquartile range, minimum and maximum values for each cluster.

r = rainy season, d = dry season. Shaded = Open Well, Unshaded = Rope-pump Well, ··· = LLOD

Microbial contamination decreased significantly (p<0.05) during the dry season for TC, E. coli, and MsC (Figure 2). Median TC contamination was 10^4.3 CFU/ 100 mL for the rainy season; in the dry season there was a half log₁₀ reduction in contamination and an overall decrease in the variability found between samples. E. coli median contamination was 10^3.2CFU/100 mL and decreased by one log₁₀into the dry season. MsC were present at 10^2.2 PFU/10 mL eluate, representing the virus recovered from one liter of filtered water.

Nascent strand RT-PCR:

Traditional methods monitoring for enteroviruses utilize a plaque assay with mammalian cells to enumerate the number of plaque forming units (PFU) in the sample; however, this method is time and labor intensive, and will often underestimate the number of viruses in environmental samples. Molecular methods, such RT-PCR, have gained popularity as a more rapid alternative to the plaque assay through targeting the enterovirus genome, but this approach cannot distinguish between infectious or inactivated viruses and may overestimate the viral threat of that sample. Integrated Cell Culture – PCR (ICC-PCR) merges the benefits of mammalian tissue culture and molecular methods, especially when coupled with a strand-specific reverse transcription reaction that targets the enterovirus’s negative RNA, since production of negative RNA is an unequivocal indicator of viral infectivity. However, strand-specific assays have been plagued with a high rate of false positives, likely due to the self-priming of RNA, mispriming in the RT step, and/or carry over of primers, enzymes, and cellular debris. Currently, there is no method capable of selectively detecting the negative RNA of Human Enterovirus B (HEV-B) species, which includes the Coxsackie and Echoviruses.

In this study, a strand-specific RT-PCR assay was developed to target an area of the 3’- non-translated region (NTR) conserved among HEV-B strains, with several modifications to increase the specificity of the reaction. HEV-B primers previously described by Oberste et al (2006) were adapted into a qPCR protocol. When using this primer set, amplification of CV and EV with a SYBR Green qPCR reaction was equally efficient at a wide gradient of annealing temperatures (Figure 3).

Figure 3. Optimization of annealing temperature. CVB4 RNA was transcribed to cDNA and amplified with a SYBR Green qPCR reaction using the HEV-B primer set. To optimize the reaction, the annealing/extension temperature ranged from 47^oC – 60^oC. The primer set amplified the cDNA at an equal efficiency across the full temperature gradient.

To develop the strand specific assay, E.coli transformed with plasmids that contained transcripts of study-strains echovirus13 (EV13) and coxsackieB4 (CVB4) were transcribed with unidirectional RNA polymerases to create stocks of positive and negative RNA. The transcribed RNA stocks were quantified, and diluted out to six point standard curve for strand-specific reverse transcription and amplification with a SYBR Green qPCR. The standard curve ranged from 0.05ng/uL – 5 x 10^-7ng/uL. Further, the R² ranged from 0.993 to 0.998 and the reaction efficiency from -2.85 to -3.35 (Figure 4).

y = -3.35x + 11.64

r² = 0.993

Figure 4. A standard curve for EV13 positive RNA.

To test for the frequency of false positives with the unmodified strand-specific assay, negative RNA was reverse transcribed to cDNA with both the forward and reverse primers, independently, and then amplified. Figure 5 demonstrates a considerable level of amplification of the reverse transcribed with the wrong RT primer, indicating a false positive due to mispriming of the primers, self-priming of the RNA, and/or other reaction artifacts. To complicate things further, the melting curve of the false positives were close to the expected melting curve of the actual PCR product (Figure 6). In our no cost extension, the strand-specific RT-PCR assay will be modified with a magnetic purification step to eliminate the false positives observed in the current protocols. Additionally, we will migrate from the current SYBR Green-based qPCR assay to one using a novel Taqman probe set targeting the 3’NTR of the HEV-B group to increase our strand specificity.

Figure 5. Evidence of false positive transcription. RNA was transcribed with the wrong RT primer amplified during the qPCR step. (a) Based on the c(t) values, the reaction efficiency of the false positives was lower than RNA transcribed with the correct RT primer. (b) The melting temperature of the false positive is often close to the melting temperature of the correct product.

(a)

Green and Yellow = True Positives Red and Blue = False Positives

(b)

Red = True Positive Tm Green = False Positive Tm

Nucleic Acid Extraction Cards:

In this year of the project, we expanded on the initial bacterial DNA extraction using the BCSI flat glass fluidic device we began in the previous year. The isolation of pure nucleic acids from samples is a crucial step in the molecular diagnosis of viral infections by nucleic acid amplification technologies (NAAT). With multiple centrifugation steps and washes, typical manual methods are often slow and limited to processing smaller sample volumes. In this year of the study, we evaluated the newly automated BCSI device for extraction of viral RNA from coliphage MS2.

The Card extraction process consisted of four steps: binding of NA, washing of the bound NA with two sequential washes, air drying of the Cards, and automated elution. Viral nucleic acids were assayed using a SYBR Green RT-qPCR assay utilizing 5 ml of each card elution. Reactions were performed using a commercial qRTPCR reagents (iScript One-Step RT-PCR Kit With SYBR Green, BioRad, Hercules, CA) in a Chromo4 RT-PCR system (BioRad, Hercules, CA) using the following temperature profile: 50C 10’, 95 C 5’, 39 cycles 95 C 15s followed by 60C for 60s. Melting curve analysis was carried out to confirm the expected amplicon.

Experiments evaluated the dynamic range of the BCSI system for phage RNA using seeded virus or viral RNA into mock eluates, transport medium and other samples. Additional experiments were performed to evaluate increasing of sample volume and to evaluate the performance in the presence of inhibiting substances, including up to 35 ng/ml of extraneous DNA. Preliminary results suggest that sample volumes of at least 4 ml may be accommodated by the BCSI device. Further initial results show a similar dynamic range, level of recovery, and purification of viral RNA is achievable with the BCSI system as compared to column based protocols.

Future Activities:

In our no-cost extension, we plan to:

Develop additional target sequences for our RPA method;
Continue nucleic acid extraction work on microfluidic cards (e.g. evaluation of different eluant types, and scaling up of volume processed);
Begin integration of filtration and nucleic acid extraction; and
Continue development of the ICC-qRT-PCR targeting the negative strand of HEV-B.

Journal Articles:

No journal articles submitted with this report: View all 16 publications for this project

Supplemental Keywords:

Virus concentration, filtration, Adenovirus, Echovirus, Coxsackie virus, Human enterovirus B, viability, ICC-qPCR, nucleic acid extraction.
, RFA, Scientific Discipline, Water, Environmental Chemistry, Drinking Water, Environmental Engineering, Environmental Monitoring, analytical methods, monitoring, pathogens, polymerase chain reaction, drinking water contaminants, drinking water monitoring

Progress and Final Reports:

Original Abstract

The 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.