Grantee Research Project Results
Final Report: Development of a Rapid, Quantitative Method for the Detection of Infective Coxsackie and Echo Viruses in Drinking Water
EPA Grant Number: R828040Title: Development of a Rapid, Quantitative Method for the Detection of Infective Coxsackie and Echo Viruses in Drinking Water
Investigators: Yates, Marylynn V. , Mulchandani, Ashok , Chen, Wilfred
Institution: University of California - Riverside
EPA Project Officer: Hahn, Intaek
Project Period: February 1, 2000 through February 1, 2002
Project Amount: $321,784
RFA: Drinking Water (1999) RFA Text | Recipients Lists
Research Category: Drinking Water , Water
Objective:
The objective of this research project was to improve the current analytical methods for quantitative detection of infective nonpolio enteroviruses (NPEVs) in drinking water. The specific objectives of this research project were to: (1) develop a molecular beacon-based (molecular beacon) reverse transcription-polymerase chain reaction (RT-PCR) method to detect NPEVs; (2) establish a potential correlation between immunomagnetic separation (IMS)-molecular beacon-RT-PCR detection and cell culture detection for infective viruses; (3) develop and evaluate real-time monitoring of virus replication in cell culture using the molecular beacon; and (4) evaluate the above methods to quantify the presence of infective NPEVs in concentrated drinking water samples.
Summary/Accomplishments (Outputs/Outcomes):
Current methods to assess the presence of infectious viruses in a sample based on cell culture techniques rely on the production of visible cytopathic effects (CPEs). Depending on the specific virus type and the concentration of viruses in the sample, it may take several days or weeks for CPEs to appear. The length of time required for the production of CPEs is one of the limitations of cell culture use for practical applications. Improved methods that can potentially be used for real-time monitoring of infective viruses are needed to enable rapid and quantitative determination.
In this project, a method that enables rapid, sensitive detection of infectious enteroviruses was developed. The method involves the use of an IMS step, which recovers intact viruses based on specific recognition of the viral capsid by, and attachment to, an antibody that is bound to a paramagnetic bead. After the viruses are separated from the sample using a magnet, the viral nucleic acid is released by heating the viruses. The viral RNA then is reverse transcribed, and the resulting cDNA is detected and quantified using real-time PCR-molecular beacon assay.
The initial RT-PCR-molecular beacon assay was designed to detect viruses in the genus Enterovirus. The assay allowed samples to be analyzed in less than 4 hours (or 8 hours when the immunomagnetic separation step is added) after RNA was extracted. This is an improvement from the conventional cell culture method, and it allowed for the detection and quantification of the viruses in real time. The assay proved to be sensitive, specific, and rapid.
The detection limit of this assay was comparable to, or better than, previous studies. When RT-PCR was integrated with the molecular beacon, the detection limit was 0.1 pfu for echovirus 11 and 1 pfu for coxsackievirus B6.
Five members of the enterovirus group and echovirus 11 were tested using the molecular beacon assay and were correctly detected. These results indicate that the molecular beacon is correctly designed to identify these viruses and also confirm the genetic relatedness of this group.
To determine the specificity of the ENTERO molecular beacon solely to echovirus 11, coxsackievirus B6, and the other members of the enterovirus group, several other microorganisms also were tested against the beacon. As expected, the organisms that tested negative were Adenovirus 2 and 15, Rotavirus, Hepatitis A virus GA76, WW#3 (Quebec), and HM175, Escherichia coli O157:H7, Salmonella typhimurium, and the bacteriophages MS2 and FX174.
The IMS-RT-PCR-molecular beacon assay was used to successfully detect low numbers of viruses in spiked environmental sample concentrates. Fewer than three viruses were detected in a surface water concentrate, although the detection limit in a groundwater concentrate was less than 1 pfu.
This IMS-RT-PCR-molecular beacon assay could prove to be a useful application to the water quality industry in assessing the potential public health risks from infection of these viruses. Molecular beacons could be used in conjunction with amplification assays to determine the efficacy of virus inactivation by disinfectants.
A previously published study suggests that real-time visualization and localization of mRNA is now possible. As few as 10 mRNA molecules could be detected using molecular beacons. Because of the exquisite sensitivity and real-time capability, one possible extension of this technology is to enable real-time detection of virus replication. By targeting the appropriate viral mRNA, it is hypothesized that it is possible to monitor the real-time progress of virus replication using molecular beacons.
In this project, in situ hybridization studies were carried out using newly synthesized viral RNA as an indicator for viral infection. A molecular beacon targeting a specific region of the enterovirus mRNA was used for the initial demonstration. Cell culture infected with enteroviruses was collected at various time points after infection and fixed. Following permeabilization, the molecular beacon was added and incubated. Whole fluorescence then was monitored with a fluorescence microscope. Cells infected for 24 hours with a high dosage of echovirus 11 were incubated with molecular beacons. Fluorescence was easily detected.
The ability of the molecular beacon to target viral RNA was tested by introducing the molecular beacon into either permeabilized infected or uninfected cells. Uninfected cells with permeabilization were not fluorescent even in the presence of the molecular beacon, whereas infected cells were brightly fluorescent, indicating that the corresponding RNA targets must be present for the spontaneous hybridization to occur.
Based on the results presented here, it is believed that the technique described easily could be adapted for other types of infectious pathogens, enabling rapid and quantitative assessment of infection.
Journal Articles on this Report : 3 Displayed | Download in RIS Format
Other project views: | All 5 publications | 3 publications in selected types | All 3 journal articles |
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Abd El Galil KH, El Sokkary MA, Kheira SM, Salazar AM, Yates MV, Chen W, Mulchandani A. Real-time nucleic acid sequence-based amplification assay for detection of hepatitis A virus. Applied and Environmental Microbiology 2005;71(11):7113-7116. |
R828040 (Final) |
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Wang A, Salazar AM, Yates MV, Mulchandani A, Chen W. Visualization and detection of infectious coxsackievirus replication using a combined cell culture-molecular beacon assay. Applied and Environmental Microbiology 2005;71(12):8397-8401. |
R828040 (Final) |
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Yeh H-Y, Yates MV, Chen W, Mulchandani A. Real-time molecular methods to detect infectious viruses. Seminars in Cell & Developmental Biology 2009;20(1):49-54. |
R828040 (Final) R833008 (Final) |
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Supplemental Keywords:
water, drinking water, exposure, pathogen, virus, microbiology, monitoring, measurement methods, reverse transcription-polymerase chain reaction, RT-PCR, molecular beacon, immunomagnetic separation, enterovirus, in situ hybridization., RFA, Scientific Discipline, Water, Environmental Chemistry, Health Risk Assessment, Environmental Microbiology, Environmental Monitoring, Drinking Water, infective coxsackie, groundwater disinfection, monitoring, detection, microbiological organisms, quantitative cell culture, exposure and effects, fluorogenic probes, exposure, community water system, echo viruses, monoclonal antibodies, analytical methods, infectious disease, treatment, microbial risk management, emerging pathogens, water quality, drinking water contaminants, drinking water treatmentProgress 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.