Grantee Research Project Results
2001 Progress 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 Period Covered by this Report: February 1, 2001 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 is to improve on the current analytical methods for quantitative detection of infective nonpolio enteroviruses (NPEV) in drinking water. The specific objectives of this research are to: (1) develop a molecular beacon-based (MB) reverse-transcription polymerase chain reaction (RT-PCR) method to detect NPEV; (2) establish a potential correlation between ion mobility spectroscopy (IMS)-MB-RT-PCR detection and cell culture detection for infective viruses; (3) using the molecular beacon, develop and evaluate real-time monitoring of virus replication in cell culture; and (4) evaluate the above methods to quantify the presence of infective NPEV in concentrated drinking water samples.
Progress Summary:
Viral Strains
The nonpolio enterovirus reference strains used for detection and optimization in these experiments were Echovirus 11 (American Type Culture Collection (ATCC VR-41 strain Gregory) and Coxsackie virus B6. A complete list of the microorganisms used in this study is found in Table 1.
Agent | Strain | Source |
Coxsackie virus B1 ATCC VR-28 | Conn-5 | ATCC* |
Cosackie virus B3 ATCC VR-30 | Nancy | ATCC |
Coxsackie B6 ATCC VR-155 | Schmitt | ATCC |
Echovirus 11 ATCC VR-41 | Gregory | ATCC |
Echovirus 17 ATCC VR-1058 | CHHE-29 | ATCC |
Echovirus 19 ATCC VR-1060 | Burke | ATCC |
Human Parechovirus 1 (formerly Echovirus 22) | Valencia | 1 |
Adenovirus 2 | sewage isolate | 1 |
Adenovirus 15 | sewage isolate | 1 |
Rotavirus | Wa | ATCC |
Hepatitis A VR-1402 | HM175 | ATCC |
Hepatitis A | WW#3 | 2 |
Hepatitis A | GA76 | 2 |
Poliovirus 1 | LscAb | ATCC |
MS2 ATCC 15597-B1 | ATCC | |
X174 ATCC 13706-B1 | ATCC | |
Escherichia coli ATCC 43895 | O157:H7 | 1 |
Salmonella typhimurium | LT2 | ATCC |
1: Los Angeles County Sanitation District, Whittier, CA; |
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2: Hepatitis Branch, National Center for Infectious Diseases, Centers for Disease Control and Prevention, (CDC) Atlanta, Georgia; ATCC = American Type Culture Collection (Manassas, VA) |
Cell Culture Conditions
The cell line used for cell culture was buffalo green monkey (BGMK). The BGMK cells were grown to confluent monolayers in culture flasks. Before the cells were infected with either CVB6 or Echo 11, they were washed twice with Tris-buffered saline solution (TBSS) prewarmed to 37°C to remove detached or dead cells and excess growth factor proteins from the cell monolayer. TBSS consisted of 0.05 Tris, 0.28 NaCl, 10 mM KCl, and 0.82 mM NaPO4. We supplemented a growth medium, 8 percent Fetal Bovine Serum (Hyclone, Inc.), to prevent toxicity. The BGMK cells were inoculated with 0.4 mL of inoculum, incubated at 37°C, and examined for cytopathic effects. The BGMK cells were inoculated with the viruses and incubated for 48 hours at 37°C in 4.0 percent CO2.
Plaque Assay
We quantified the viruses using the plaque assay method, which included distributing tenfold serial dilutions of virus stock on confluent cells in six well plates and incubating for 60 minutes at 37°C in 4.0 percent CO2. A maintenance media of 1.5 percent agar overlay was applied at 37°C in 4.0 percent CO2 during the 48-hour incubation period. Plaques were visualized after overlay removal, fixation of cells with ethanol, and stained with 8.0 percent w/v crystal violet and 20 percent v/v 95 percent ethanol in deionized water.
RNA Extraction
We extracted Echo 11 and CVB6 RNA using the phenol/chloroform method. We transferred the viruses from cell culture to microcentrifuge tubes in 100 ml aliquots. To the 100 µl of cell culture fluid infected with Echo 11, 100 µl of phenol/chloroform was added. It was vigorously vortexed for 5 minutes to denature proteins. We centrifuged mixture for 5 minutes at 10,000 rpm to separate the aqueous and organic phases. We transferred the top aqueous layer phase to a new microcentrifuge tube, avoiding the white interface protein layer. Phenol/chloroform was added again, vortexed, and centrifuged until the white protein layer was gone, and transferred to a new tube. Afterwards, 200 µl of a mixture of 4.0 M LiCl and 100 percent ice-cold ethanol was added to the aqueous layer and vortexed gently. It was stored at -20°C overnight. We centrifuged the samples the next day at 14,000 rpm for 10 minutes. Two-hundred and fifty microliters of ice-cold 70 percent ethanol was added, vortexed gently, and stored at -20°C overnight. The next day, the tubes were centrifuged at 14,000 rpm for 10 minutes. The ethanol was extracted, leaving the clear pellet in the tube until all ethanol had evaporated. Subsequently, the extracted RNA was resuspended in 30 µl of Tris-EDTA (TE) buffer, pH 7.8.
Primer and Molecular Beacon Design
Primer Design. We developed the following primers for the detection of all human enteroviruses, including Echovirus 11 using the highly conserved 5' noncoding region. The following primers were slightly adjusted from the primer sequences used by Abbaszadegan, et al. (1993). The upstream primer, base pairs (BP) 447 to 466 (ENTERO1F: 5'-TCC TCC GGC CCC TGA ATG CG-3') was synthesized sense to genomic RNA and had a Tm of 77.4°C. The downstream primer, BP 582 to 601 (ENTERO2R 5'-ATT GTC ACC ATA AGC AGC CA-3'), was synthesized antisense to genomic viral RNA and had a Tm of 63.0°C. We designed the primer pair to amplify a 155-BP fragment of RNA of the 5' noncoding region. The primers were synthesized by Genosys (Fisher Scientific). The primers were resuspended in autoclaved double-deionized water and stored at -20°C in 20 µM aliquots.
Molecular Beacon Design. The antisense 25-BP probe moiety (ENTERO MB: 6-FAM-5'-cgagcg GCA GCG GAA CCG ACT ACT TTG GGT G cgctcg-3'-DABCYL) was designed to complementarily target the internal region of the PCR amplicon and is flanked by two 6-BP arms (underlined). The beacon was designed to identify the 5' noncoding region of RNA of the enterovirus group and to allow for two mismatches in its complementary sequence to account for the large diversity found in the Enterovirus genus. Echovirus 11 and Coxsackie virus B6 will be the representatives for the genus. We purchased the beacon from the Midland Certified Reagent Company (Midland, TX). We synthesized the molecular beacons using oligonucleotides containing sulfhydryl groups at the 5' ends and primary amino groups at the 3' ends. The fluorophores were then attached to the sulfhydryl groups and the quenchers were coupled to the amino groups. The 5' end was labeled with the fluorophore, 6-FAM (6-carboxyfluorescein), which has an excitation of 488 nm and an emission of 518 nm. The 3' end was labeled with the quencher DABCYL (4-[4'-dimethylaminophenylazo]benzoic acid). The probe was resuspended in autoclaved double-deionized water, wrapped in aluminum foil for protection from light, and stored at -20°C in 76 µM aliquots.
Signal-to-Background Ratio Determination and Thermal Denaturing Profile. The signal-to-background ratio for the ENTERO MB was calculated in the following manner:
(Fprobe-target - Fbuffer)/(Fhairpin - Fbuffer).
To determine the optimal temperature for hybridizing the MB to the amplicon and the recording of fluorescence, we performed a thermal denaturation profile. We recorded fluorescence on varying amounts of MgCl2, 0.39 µM of the probe, and with or without 2.5 µM of the probe's complementary oligonucleotide. We recorded the fluorescence intensity for the varying mixtures, which were heated to 98°C and decreased 1°C every minute until it reached 10°C, using the Bio-Rad iCycler iQ Real-Time PCR Detection System (Bio-Rad, Hercules, CA).
RT-PCR Conditions and Detection Limit Determinations. Because it is necessary for RNA to be reversely transcribed before being amplified in PCR, we used the Bio-Rad iCycler iQ Real-Time PCR Detection System (Hercules, CA) to perform the RT. The reagents used were from the GeneAmp® RNA PCR Core Kit from Applied Biosystems (Foster City, CA) and their concentrations were calculated from a final volume of 100 µl. For the RT, the following reagents were added to Bio-Rad's 200 µl thin wall PCR plate: 0.3X PCR Buffer II, 0.5 µM of random hexamers, and 250 µM of each deoxyribonucleoside triphosphate. The plate was heated to 95°C for 5 minutes to denature 10 µl of RNA in addition to minimizing secondary structures. We added 50U of Murine Leukemia Virus (MuLV) reverse transcriptase, 20U of RNase inhibitor, and 1.5 µM MgCl2 to the reaction mixture; we performed RT under the following conditions: 25°C for 10 minutes, 42°C for 60 minutes, and a final 100°C for 5 minutes to denature the reverse transcriptase. We used reagents from the GeneAmpâ RNA PCR Core Kit from Applied Biosystems to prepare master mixes for the experiments. For the real-time PCR assay, we added following reagents to Bio-Rad's 200 µl thin wall PCR plate after the RT reaction: 0.7X PCR Buffer II, 1µM MgCl2, 0.1 µM of forward and reverse primers, 2.5U of Taq polymerase (Promega, Madison, Wisconsin), 0.39 µM of molecular beacon, and autoclaved double-deionized water was added for a final volume of 100 µl. We sealed each well with optical quality sealing tape (Bio-Rad, Hercules, CA). We performed the thermal cycling conditions as follows: denaturation for 30 seconds at 95°C, annealing of primers and MB for 1 minute at 50°C, and extension for 30 seconds at 72°C for 40 cycles. We recorded fluorescent intensity data at each step of the PCR assay. However, we programmed the real-time amplification plots to be observed in real-time during the annealing step. At the end of the 40 cycles, the program automatically analyzed the data.
To perform the detection limit studies, tenfold serial dilutions of the viral RNA were made in autoclaved double-deionized water. The number of RNA molecules were calculated by plaque assay. For each experiment, a no-template control was used. This negative control substituted autoclaved double-deionized water for RNA template.
Results
Signal-to-Background Ratio and Thermal Denaturation Profile of Molecular Beacon. Using the formula, (Fprobe-target - Fbuffer)/(Fhairpin - Fbuffer), the signal-to-background ratio was calculated to be 48.47. The thermal denaturation profile was performed for three purposes: (1) to determine stability of the hybrid between the amplified target and the molecular beacon probe; (2) to determine the optimal annealing temperature for the probe; and (3) to determine the optimal MgCl2 concentration, which would give the greatest fluorescence intensity between the beacon-target hybrid. To ascertain the optimal recording temperature for the real-time RT-PCR assay, we investigated the thermal denaturing profile of the beacon in the presence or absence of a perfectly complementary target oligonucleotide using the iCycler. Figure 1 demonstrates the fluorescence emission due to the conformational change of the molecular beacon as a function of temperature. The profile confirms that at low temperatures, the arms of the MB form a hairpin structure, therefore inhibiting fluorescence. As the temperature increases to 36°C, we observe the maximum difference in the signal-to-background ratio. At 60°C, the probe-target hybrid begins to denature and assumes a random coil configuration and we observe a decrease in fluorescence. The profile indicates that an optimal fluorescence recording temperature of ENTERO MB should be below 60°C.
Figure 1. Thermal Denaturation Profile of the Enterovirus Molecular Beacon. The conformational change of the molecular beacon is dependent on temperature. In the presence of
Its complementary target oligo (), the conformational change of the molecular beacon is observed as an increase or decrease in fluorescence. When the target-beacon hybrid is formed, the greatest intensity of fluorescence is observed. In the absence of its complementary oligo (), there is no significant change in fluorescence and the beacon remains in the hairpin structure.
Detection of Echo 11 and CVB6 With Real-Time RT-PCR-MB Assay. We conducted investigations into the probe's ability to detect Echo 11 and CVB6 by real-time RT-PCR assays. We designed primers to specifically amplify a section of the 5' noncoding region of the Enterovirus group with Echo 11 and CVB6 being its representatives. As expected, the primers amplified the 155-bp region specified for Echo 11 and CVB6, and the MB detected both (see Figures 3 and 4). Subsequently, we used this RT-PCR-MB assay for sensitivity and specificity studies.
Detection of Other Microorganisms Using the Molecular Beacon. We conducted experiments to determine the molecular beacon's ability to detect additional members of the Enterovirus genus and other potential microorganisms found in water. Adenovirus 2 and 15, Rotavirus, Hepatitis A virus (HAV) GA76 and WW#3 (Quebec), E. coli O157:H7, S. typhimurium, and the bacteriophages MS2 and X174 were negative when tested in the RT-PCR-MB assay. These results are summarized in Table 2.
Agent | Strain | Detection |
Coxsackie virus B1 ATCC VR-28 | Conn-5 | + |
Cosackie virus B3 ATCC VR-30 | Nancy | + |
Coxsackie B6 ATCC VR-155 | Schmitt | + |
Echovirus 11 ATCC VR-41 | Gregory | + |
Echovirus 17 ATCC VR-1058 | CHHE-29 | + |
Echovirus 19 ATCC VR-1060 | Burke | + |
Human Parechovirus 1 (formerly Echovirus 22) | Valencia | - |
Adenovirus 2 | sewage isolate | - |
Adenovirus 15 | sewage isolate | - |
Rotavirus | Wa | - |
Hepatitis A VR-1402 | HM175 | - |
Hepatitis A | WW#3 | - |
Hepatitis A | GA76 | - |
Poliovirus 1 | LscAb | + |
MS2 ATCC 15597-B1 | - | |
X174 ATCC 13706-B1 | - | |
E. coli ATCC 43895 | O157:H7 | - |
S. typhimurium | LT2 | - |
We observed that the RT-PCR assay that we developed could be used to amplify other viruses. Therefore, specificity of the RT-PCR-MB assay to the Echo 11 and the enteroviruses needed to be demonstrated. Two viruses that could be amplified using the same RT-PCR assay were HAV HM175 and Poliovirus 1. Using the MB with RT-PCR, HAV HM175 was not detected, amplified, or visualized by gel electrophoresis. However, the assay detected and amplified Poliovirus 1. This was later confirmed by gel electrophoresis.
Real-Time RT-PCR-MB Sensitivity. The sensitivity of the real-time RT-PCR reaction was determined using tenfold serial dilutions of Echo 11 and CBV6 RNA transcripts containing 0.1, 1, 10, 100, and 1,000 RNA molecules. Any fluorescent signal that was tenfold higher than the standard deviation of the mean baseline emission was indicative of a positive detection. We evaluated variations in sample preparation of Echo 11 in five separate sets of real-time RT-PCR assays using the aforementioned number of RNA molecules. A comparison of the critical (Ct) values of the five different sets shows little variability between the assays (see Table 3). As shown in Figures 2 and 3, the detection limit of CVB6 was 1 pfu; tenfold lower than Echo 11.
Days | |||||||
pfu's | 1 | 2 | 3 | 4 | 5 | Mean | S.D. |
1,000 | 24.705 | 25.55 | 24.546 | 24.712 | 24.147 | 24.732 | 0.511633 |
100 | 27.326 | 27.148 | 27.836 | 28.442 | 27.467 | 27.6438 | 0.512869 |
10 | 30.937 | 31.019 | 31.328 | 32.113 | 30.993 | 31.278 | 0.490997 |
1 | 34.51 | 33.446 | 34.744 | 35.563 | 33.694 | 34.3914 | 0.850175 |
0.1 | 36.548 | 35.11 | 36.511 | 36.981 | 37.486 | 36.5272 | 0.885209 |
Figure 2. A Detection Limit of 0.1 pfu was Achieved With Echo 11. () 1000 pfu, () 100 pfu, () 10 pfu, () 1pfu, () 0.1 pfu, () Negative Control
Figure 3. A Detection Limit of 1 pfu was Achieved for Coxsackie Virus B6. () 1000 pfu, () 100 pfu, () 10 pfu, () 1 pfu, (+) negative control.
Discussion
The RT-PCR-MB assay that we developed was designed to detect the genus we extracted from the Enterovirus. The assay allowed samples to be analyzed in less than 4 hours after RNA. This is an improvement from the conventional cell culture method and 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 or better than previous studies. When we integrated RT-PCR with the molecular beacon, the detection limit was 0.1 pfu for Echo 11 and 1 pfu for CVB6. When the amplified fragments were visualized on ethidium bromide-stained 2.5 percent agarose gel, the band for 0.1 pfu was not visible. One possibility for less than 1 pfu detection of Echo 11 may have been due to contamination of RNA molecules and pipetting errors. However, much of it was later avoided with the use of master mixes to perform RT-PCR and aliquoting the RT-PCR reagents. Aliquoting facilitated by decreasing the probability of contamination to the other reagents. Meanwhile, the master mixes allowed for consistency in the concentrations of reagents throughout the reaction and reduced the risk for contamination. Also, cell culture techniques can only detect viruses that are infectious. RT-PCR cannot distinguish infectious viruses from noninfectious viruses or detect the presence of intact virus particles (it is possible that "naked" RNA could be detected in an assay). There may have been naked RNA in cell culture that was not detected, because the virus cannot replicate and produce CPE. Therefore, we extracted the naked RNA with the RNA from intact viruses. It was possible that this is the reason we see such a low detection limit. A confirmation of the detection of 0.1 pfu might be possible if the number of cycles was extended so that amplification of this amount is possible. However, this would increase the amount of time required for PCR, thereby making the detection and quantification much slower. Also, the 0.1 pfu detection limit was achieved on 5 separate days and the standard deviation was less than one cycle, making this 0.1 pfu detection limit reliable.
We correctly detected five members of the Enterovirus group and Echo 11. These results indicate that not only was the MB correctly designed to identify these viruses, but it also confirms the genetic relatedness of this group. However, Echovirus 22 (Echo 22) was not identified by the ENTERO MB. This may be due to the studies conducted on reclassifying Echo 22 as Human Parechovirus type 1. Several studies have demonstrated that Echo 22 is not a typical enterovirus. Though the genome organization of Echo 22 is similar to others in the Picornavirus family, the amino acid identities of polypeptides with the corresponding proteins of other picornaviruses are only in the 14-35 percent range. Consequently, Echo 22 has been classified as the member of a sixth genus, Parechovirus and has been renamed Human Parechovirus type 1 (HPeV1).
To determine the specificity of the ENTERO MB solely to Echo 11, CVB6, 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 (HAV) GA76, WW#3 (Quebec), and HM175, E. coli O157:H7, S. typhimurium, and the bacteriophages MS2 and X174. Yet, Poliovirus 1 was detected using this developed assay, even though this assay was developed to detect NPEV. A summary of these results is shown in Table 3. This is because Polioviruses and Coxsackie B viruses have been shown to be quite homogeneous and are relatively related. Because the ENTERO MB was designed to identify Coxsackie B viruses, it is not surprising that it was also able to detect polio.
The detection phenomenon, known as the "hook effect," occurred in the assays (see Figure 3). It is caused by the competition between the reassociation of single strands of PCR product after denaturation and the binding of the hybridization probes to target in this late PCR phase. Because the PCR process amplifies both template strands and the amounts of PCR products are high, the two strands reassociate faster than the hybridization probes can bind to their target (Roche Molecular Biochemicals).
This RT-PCR-MB 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 gather occurrence data in drinking water, assess the vunerability of groundwater to surface contamination, and to determine the efficacy of virus inactivation by disinfectants.
Future Activities:
We will continue to: (1) develop a MB RT-PCR method to detect NPEV; (2) establish a potential correlation between IMS-MB-RT-PCR detection and cell culture detection for infective viruses; (3) using the MB, develop and evaluate real-time monitoring of virus replication in cell culture; and (4) evaluate the above methods to quantify the presence of infective NPEV in concentrated drinking water samples.
Journal Articles:
No journal articles submitted with this report: View all 5 publications for this projectSupplemental Keywords:
drinking water, groundwater, pathogens, microbiology, monitoring, measurement methods., 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.