Grantee Research Project Results
Final Report: A Novel Molecular-Based Approach for Broad Detection of Viable Pathogens in Drinking Water
EPA Grant Number: R833011Title: 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 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.
Summary/Accomplishments (Outputs/Outcomes):
In this final year of the project, our work focused on:
- Continued development of our ratiometric pre-rRNA method
- Nucleic acid extraction from filter concentrates using microfluidic cards
- Continued development of nascent strand RT-PCR method for Human Enterovirus B.
Work Progress:
pre-rRNA RT-PCR:
In this year of the project, we filed a patent on our ratiometric pre-rRNA method (International Patent Number PCT/US09/67565 titled RATIOMETRIC PRE-RNA analysis).
Nucleic acid extraction:
In this year of the study, we continued to evaluate a novel macrofluidic flat glass card system, able to process larger sample volumes than standard column RNA extraction kits. Virocap disposable capsule filters were used to concentrate 10-200 L volumes of surface, tap, and groundwater. The filters were eluted with 1.5% beef extract (0.05M glycine 0.1% Tween 80) or Optima RE elution buffers. Eluates from the various water concentrates were spiked with bacteriophage MS2 (a RNA virus similar in size, persistence, and survival characteristics to many waterborne enteric viruses of public health concern). Samples were lysed and bound to flat glass cards, which were then extracted using an automated system; eluates were recovered and stored for subsequent analysis. MS2 recovery was compared between PBS, elution buffers, and water concentrates eluted by each elution buffer. Four MS2 concentration levels were evaluated for each sample type, and each sample was run in duplicate on individual glass cards. MS2 was quantified using a TaqMan one-step qRT-PCR reaction.
In experiments comparing seeded eluant controls to seeded PBS, minimal C(t) shifts were observed for recovery of MS2 in PBS vs the beef extract eluant control; R2 and reaction efficiency remained comparable.
Two experiments were conducted using surface and tap water eluted using beef extract, two experiments were conducted using surface and tap water eluted using Optima RE, and three experiments were conducted using 100 L and 200 L groundwater eluted with Optima RE. A slight shift in the standard curve was observed between the beef extract eluant control and water concentrates. However, replicates demonstrated low variability between cards and only small deviations of C(T) despite interference. Surface water eluted into beef extract demonstrated greater inhibition in both experiments conducted compared to other water types. However overall, the difference in card extraction efficiency between water sample types was minimal, and R2 and qPCR reaction efficiencies were comparable (R2 > 0.98 and 94-99% reaction efficiencies).
The two experiments using surface and tap water eluted with optima had R2 values >.93 with the exception of optima itself, which demonstrated an R2 value of .86 and .92 in the two experiments. This may have been due to an error in the functioning of the machine; thus, the two experiments were excluded from further consideration in the data set. In general, the results from both experiments using optima produced similar results to the experiments with beef extract elution. Experiments using groundwater at 100 L and 200 L eluted into optima showed the strongest results in terms of replication.
Three continuous flow experiments were conducted to test if larger samples could be processed on the cards. In experiments, 2 mL and 4 mL volumes were compared to the standard volume of ~1 mL, resulting in equal or better recoveries observed for the standard volume.
Nascent strand RT-PCR:
In the past year, we continued development of a nascent strand RT-PCR method targeting echovirus (EV) and coxsackie virus (CV), members of a newly reorganized group of human enteroviruses called Human Enterovirus B (HEV-B). Our Integrated Cell Culture RT-PCR (ICC-PCR) method targeting the negative (replicative) strand of HEV-B viruses merges the benefits of mammalian tissue culture and molecular methods.
Previously, we demonstrated false positive amplification of negative strand RNA using our SYBR green assay. Subsequently, we have developed a Taqman-based probe and integrated a magnetic purification step to increase strand specificity.
Sequences from 56 HEV-B serotypes, representing almost all known serotypes plus several strains, were imported into Geneious Pro v. 4.8.5. (Biomatters Ltd., Auckland, NZ) and the 3'NTR, composed of 102-109 base pairs, was aligned. A degenerate, dual labeled probe (HEVB-451b) was designed around existing primers targeting the HEV-B 3'NTR region (HEVB-481F/424R primer set; Oberste et al., 2006). The probe was designed to include as many HEV-B serotypes as possible, but with a preference for the 15n responsible for 83.5% of all enteroviral infections in the United States (Khetsuriani et al., 2006). The sensitivity of the HEV-B primer/probe set was evaluated with an 8-point standard curve of a serially10-fold diluted plasmid stock, previously confirmed as having the HEV-B 3'NTR insert. The standard curve ranged from 34 to 3.4 x 108 theoretical copy numbers. The performance of the probe was assessed with 11 HEV-B serotypes, including nine of the top 10 serotypes involved in enteroviral infections from 1970-2005. RNA was extracted from the virus stocks, transcribed to cDNA with HEV-B specific primers, and amplified with the qPCR protocol developed for the HEV-B primer/probe set. RNA from Poliovirus Type 1 (Vaccine strain LSc) was included in the evaluation as a negative control to test for cross-reactivity with non-HEV- B enteroviruses. All 11 HEV-B serotypes tested for specificity were detected by the HEV-B primer/probe set. Poliovirus, Type 1, a closely related HEV-C serotype used as a negative control, was not detected.
To evaluate whether biotin labeled primers, which are needed to facilitate magnetic purification of the negative strand, affect the performance of the HEV-B primer probe set, a biotin label was added to the 5' end of the HEV-B 481F primer and used in synthesis of cDNA from negative sense RNA. The correlation and slope of the standard curve were used as markers to compare the robustness and efficiency of the biotinylated and non-biotinylated assays. Similarly, sensitivity between the two primers was compared. The modified reverse transcription primer did not affect the reactions. The slopes of the biotinylated and non-biotinylated reactions were 3.51 and 3.90, respectively; further, the r2 for both reactions was 0.99.
Dynabeads (Invitrogen, Carlsbad, CA, USA) are streptavidin-coated magnetic beads used to separate out biotinylated molecules. A number of variables were optimized to increase cDNA recovery in magnetic purification. The cDNA recovery was assessed by comparing Ct values of unpurified cDNA with purified cDNA. Larger differences in Ct values between the two reactions indicated a less efficient recovery. When necessary, the percent recovery also was calculated: the quantity of the purified cDNA was extrapolated from the standard curve and compared to the known quantity of the unpurified cDNA.
Once the protocol was developed, (-)RNA was diluted along a 10-fold standard curve, transcribed to cDNA with a biotinylated HEV-B 481F primer (as described in 2.3.2.), and a portion was purified using the optimized method. The purified and unpurified samples were amplified with qPCR to assess any effects that the Dynabead-complex may have had on the efficiency and sensitivity of the assay. Differences in Ct values between the two sample-types were used to develop and optimize the magnetic purification protocol, with the smallest difference in Ct values indicating the best cDNA recovery. The properties of Dynabeads vary based on their application and the characteristics of the biotinylated compound. Of the four kits tested, the M-270 had the most consistent recovery over a range of cDNA concentrations. This was noted over a number of magnetic purification trials. Once the magnetic purification protocol was developed, the assay was evaluated for any effect that the Dynabead-complex may have had on the subsequent qPCR assays. In addition, the percent recovery of the cDNA was calculated as an additional index for the robustness of the method. The purified and unpurified standard curves had similar slopes, at -3.20 and -3.24, respectively. Further, the r2 for both reactions were excellent, indicating that the Dynabead-complex did not interfere with the amplification of the cDNA, even at the lowest concentrations.
Until this point, the development of the strand-specific assay was based on (-) RNA transcripts, an artificial template compared to the goal of detecting (-)RNA from within host cells infected with a HEV-B virus. To evaluate the assay's abilities in a more realistic environment, an integrated cell culture (ICC) step was designed to precede the strand-specific qPCR. A short-course ICC-assay was set up to determine how quickly HEV-B species begin replicating, as well as an earliest time-point for detection of (-)RNA. The virus inoculum per dish was 500,000 PFU, 5,000 PFU, or 500 PFU, and one dish was inoculated for each time point at each virus titer. Cells were incubated at 37 oC for 45 minutes, rocking frequently. Cells were harvested at the following post-infection time points: 1.5 hours, 2 hours, 2.5 hours, 3 hours, and 5 hours. Viral RNA was extracted from the cells using the Qiagen QIAamp viral RNA mini kit and then reverse transcribed using the biotinylated HEVB-481F primer. A portion of the cDNA was magnetically purified, and a qPCR assay with the HEV-B primer/probe set was carried out with purified and unpurified cDNA.
During the short-course ICC-step for CVB5, (-)RNA was detected for all three titers at 5 hours post infection (h.p.i.), indicating replication of the virus within the infected cells. Negative sense RNA was not detected before 5 h.p.i. The concentration of (-)RNA was dependent on the titer of the virus inoculum, as indicated by decreasing Ct values with increasing RNA concentration. Further, there was a minimal loss in cDNA during magnetic purification, with Ct values between the purified and unpurified cDNA ranging between 0.4 and 1.3 cycles, comparable to the difference in Ct values observed with the artificial (-)RNA transcripts. The initial inoculation titers of 500, 5000, and 500,000 PFU of CVB5 corresponded to 0.5, 5, and 500 PFUs per qPCR reaction.
Journal Articles on this Report : 4 Displayed | Download in RIS Format
Other project views: | All 16 publications | 4 publications in selected types | All 4 journal articles |
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Bennett HB, Shantz A, Shin G, Sampson ML, Meschke JS. Characterization of the water quality from open and rope-pump shallow wells in rural Cambodia. Water Science and Technology 2010;61(2):473-479. |
R833011 (Final) |
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Bennett HB, O'Dell HD, Norton G, Shin G, Hsu F-C, Meschke JS. Evaluation of a novel electropositive filter for the concentration of viruses from diverse water matrices. Water Science and Technology 2010;61(2):317-322. |
R833011 (Final) |
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Cangelosi GA, Weigel KM, Lefthand-Begay C, Meschke JS. Molecular detection of viable bacterial pathogens in water by ratiometric pre-rRNA analysis. Applied and Environmental Microbiology 2010;76(3):960-962. |
R833011 (Final) |
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Parker JK, Chang T-Y, Meschke JS. Amplification of viral RNA from drinking water using Transplex™ whole transcriptome amplification. Journal of Applied Microbiology 2011;111(1):216-223. |
R833011 (Final) |
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Supplemental Keywords:
RFA, Scientific Discipline, Water, Environmental Chemistry, Environmental Monitoring, Drinking Water, Environmental Engineering, monitoring, pathogens, biomarkers, drinking water monitoring, polymerase chain reaction, analytical methodsProgress and Final Reports:
Original AbstractThe 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.