Integrated Cell CulturePCR for Detection of Enteric Viruses in Environmental Samples

Kelly A. Reynolds

1. Introduction

Recently, an integrated cell culture/polymerase chain reaction (ICC/PCR) technique has been developed for the detection of viruses in environmental samples providing a reliable method for practical analysis and direct monitoring of environmental samples for viral pathogens (1,2). CC/PCR allows for detection of infectious viruses in hours to days compared with the days or weeks necessary with cell culture alone. Bacterial indicator organisms are commonly used to evaluate environmental samples with respect to fecal contamination and potential public health impacts. These organisms do not correlate well with the presence of viruses, but a rapid, reliable method was not previously available for direct virus testing. Using ICC/PCR, environmental samples may be directly surveyed for pathogenic viruses, in a timely manner. Direct virus analysis will lead to better assessment of the presence and risk of human enteric viruses in the environment, so that control measures may be developed with true virus occurrence data. The ICC/PCR approach combines two previously applied virus detection methods, conventional cell culture and PCR amplification, utilizing the major advantages and overcoming the major limitations of each methodology when used alone.

Cell culture assay is the standard method for the detection of viable human viruses (i.e., poliovirus, coxsackievirus, echovirus, adenovirus, hepatitis A virus, reovirus, and rotavirus) in environmental samples, serving as the method against which all newer technologies are evaluated. Although cell culture is theoretically capable of detecting a single viable virus in relatively large volumes of sample, the time required for confirmed results with conventional cell culture makes it an impractical method for routine monitoring of environmental samples. Furthermore, cell culture does not detect noncytopathogenic viruses (viruses that are viable, infecting cells, and continually spreading to neighboring cells but that do not cause a visible cytopathogenic effect [CPE] on the cell monolayer). Rotavirus and most wild-type hepatitis A viruses (HAV) are infectious to cell cultures but do not produce a clear CPE.

Another disadvantage to conventional cell culture is that a single cell line may support the growth of a variety of different viruses, preventing specific identification of the viruses present and allowing less fastidious viruses to proliferate, masking the presence of other, potentially pathogenic, viruses. Finally, conventional cell culture is subject to toxicity, meaning that substances such as toxic chemicals, waste products, suspended particles, and humic and fulvic acids can inactivate the cell monolayer. The presence of bacteria and fungi may also inactivate the cell monolayer, prior to virus infection. Cell culture toxins are common to environmental samples and visually mimic viral CPE, leading to false-positive results. Toxic samples must be purified, risking loss of original virus populations, and reassayed, at an increased cost of analysis.

Molecular methods such as PCR were developed to detect a wide range of viruses directly, including noncytopathogenic strains, and they have been used extensively for the sensitive detection and identification of viruses in clinical and environmental samples. The use of specifically designed nucleotide primers allows the sequences of target viruses to be amplified rapidly and specifically. Definitive results are available in less than 1-2 d.

There are some major disadvantages, however, to direct PCR amplification of viruses from environmental samples, including: (1) the presence of PCR-inhibitory compounds in sample concentrates; (2) the small reaction volumes; and (3) the detection of noninfectious virus particles. PCR-inhibitory compounds are naturally present in the environment (i.e., humic and fulvic acids, proteins, metals, and salts) and, when present, prevent reaction enzymes from amplifying a target sequence, producing a false-negative result. PCR inhibition can be very difficult, if not impossible, to overcome without loss of the target viral sequence. In addition, owing to the expense of the PCR reagents and the limitations of the thermal cycling equipment currently available, only small reaction volumes (approx 10 ^L/reaction) can be examined by PCR.

Finally, PCR cannot distinguish between infectious and noninfectious viral targets. A commonly observed phenomenon with viruses is the presence of a particle-to-plaque-forming unit (PFU) ratio. A plaque-forming unit possesses the nucleic acid, capsid, and receptor components and is able to infect host cells; however, viral nucleic acid can be present in the absence of a complete, infectious virus. Depending on the environment, the particle/PFU ratio may be as high as 1000. Virus cultures grown under low-stress laboratory conditions typically have a particle/PFU ratio of 10. PCR does not differentiate between noninfectious and infectious viruses and thus expectedly overestimates the risk of virus infection in exposed populations. Since the actual ratio of particles to PFU is not known for individual environments, the level of overestima-tion of risk cannot be easily determined. Detection of noninfectious particles is particularly important in treated water supplies, in which viruses originally present are rendered noninfectious by the treatment process, such as chlorine disinfection, while their nucleic acid sequences persist. These particles are detectable by PCR but are not expected to be a public health risk.

The ICC/PCR method combines both cultural and molecular techniques for rapid detection of viable human viruses and thus is able to detect viruses in large equivalent volume concentrates, without the limitations of cell culture toxicity or PCR inhibition.

Inhibition is inherently overcome by the dilution of the sample concentrate with cell culture media. In addition, viruses are grown to much higher numbers in the cell culture flask, further ensuring the success of the PCR amplification. Therefore, using ICC/PCR, a large population of viruses is being detected with a low concentration of inhibitors, eliminating the uncertainty of true vs false negative results with direct PCR alone. Cell culture toxicity is minimized since the assays can be stopped and viruses detected prior to cell death owing to toxicity, eliminating the uncertainty of true vs false positive results with conventional cell culture alone.

Previous studies have shown the effective application of ICC/PCR methodology on environmental sample concentrates that were inhibitory to direct PCR (1,2) and at virus concentrations as low as 0.001 most probable number (MPN) per liter of original sample concentrate (3). The ICC/PCR method was able to detect enterovirus concentrations of >10 PFU per cell culture flask inoculum, after only 5 h of incubation. Detection of 1 PFU/flask occurred after only 20 h of incubation, compared with a minimum of 5-7 d using conventional cell culture alone (2). Finally, ICC/PCR has been used to evaluate the effectiveness of virus inactivation protocols (i.e., chlorine disinfection) (4). Compared with results from single-passage cell culture, ICC/PCR determined that the contact time for chlorine disinfection of poliovirus is up to five times greater than previously thought.

The primary limitation of the ICC/PCR procedure is that both cell culture and molecular laboratories must be maintained and viruses must be culturable to be detected. Some of the more significant public health viruses, i.e., Norwalk agent and enteric caliciviruses, cannot be grown in conventional cell culture, leaving direct molecular methods one of the only currently available options for their detection in environmental samples.

2. Materials

2.1. Cell Culture

Virus stocks, cell cultures, and environmental sample concentrates should be considered positive for human pathogenic viruses, handled with gloves, and contained in a biosafety level 2 (BL2) laboratory, or higher, equipped with appropriate BL2 safety equipment.

1. Virus control stocks (i.e., poliovirus strain LSc-2ab).

3. Sample concentrate (stored at -70°C until use).

4. Cell culture maintenance medium with serum warmed to 37°C (i.e., minimum essential medium [MEM]/L-15 with 2% fetal bovine serum).

5. Inverted microscope (100X magnification).

2.2. RT-PCR Amplification

1. 10X PCR buffer: 500 mM potassium chloride, 100 mM Tris-HCl, pH 8.3. Store at -20°C, and thaw just prior to use.

2. 25 mM MgCl2. Store at -20°C, and thaw just prior to use.

3. 10 mM Deoxynucleoside triphosphates (dNTPs). Combine all four dNTPs in one master mix, store at -20°C, and thaw just prior to use.

4. Mineral oil, PCR grade.

5. Thermal cycler (Perkin Elmer; Applied Biosystems, Forest City, CA).

6. Random hexamers. Store at -20°C, thaw on ice just prior to use, and keep on ice at all times.

7. Avian myeloblastosis virus reverse transcriptase (AMV-RT; Life Sciences, St. Petersburg, FL). Store at -20°C, and keep on ice at all times.

8. Ribonuclease inhibitor (RNasin; Promega, Madison, WI). Store at -20°C, and keep on ice at all times.

9. Upstream, downstream, and internal primer set. Store at -20°C, and thaw just prior to use. Specific primers, used to amplify a 149- and 192-bp target sequence on enteroviruses, include a downstream enterovirus primer, nucleotides 577-594: 5'-TGT CAC CAT AAG CAG CC-3'; with an upstream enterovirus primer, nucleotides 445-465: 5'-TCC GGC CCC TGA ATG CGG CT-3' (5). The seminested primer used to yield a 105-bp product for enteroviruses is an internal downstream enterovirus primer from nucleotides 530-550: 5'-CCCAAAGTAGTCGGTTCCGC-3') (2).

10. High-performance liquid chromatography (HPLC) grade sterile water. Store at -20°C, and thaw just prior to use.

11. AmpliTaq and Amplitaq gold enzymes (Applied Biosystems). Store at -20°C, and keep on ice at all times.

12. Metaphor agarose (FMC Bioproducts, Rockland, ME).

13. Loading buffer: 20% ficoll, 1% sodium dodecyl sulfate, 0.25% bromphenol blue, 0.1 M disodium EDTA, pH 8.0. Store at room temperature.

14. Gel electrophoresis power source, gel trays, and buffer trays, and recirculating pump with cooling ice bath.

15. TAE buffer (1% Tris-acetate/EDTA). Store at room temperature, or at 4°C.

16. Ethidium bromide solution (0.5 ^g/mL). Store at room temperature, shielded from light. Prepare new solution every five staining cycles. Ethidium bromide is a known mutagen. Always wear gloves when handling.

17. 123-bp Ladder (Gibco BRL, Gaithersburg, MD).

18. 302-nm UV-transilluminator. Wear safety goggles and shield exposed skin when using UV light source.

3. Methods

Methods for the filtration, elution, and concentration of viruses from a variety of environmental samples including water, sewage, sludge, and soil have been reviewed elsewhere (6,7). The following sections detail protocols specific to the ICC/PCR method of enteric virus detection.

3.1. Cell Culture

Various cell types may be used for the isolation of enteric viruses (see Note 1). A common continuous cell line, known as BGM, derived from African green monkey kidneys, is highly susceptible to enteroviruses (polioviruses, coxsackieviruses, and echoviruses) and is easily grown in culture flasks. A detailed protocol for the growth, maintenance and quality control requirements of BGM cells is supplied by the US Environmental Protection Agency (8).

1. Briefly, cells are grown into confluent monolayers in tissue culture grade plastic, or glass vessels, bathed in warmed (37°C) MEM/L-15 (equal parts Eagle's MEM/

Leibovitz medium) supplemented with 10% (v/v) fetal bovine serum, 7.5% (w/v) stock sodium bicarbonate solution, and antibiotics (100 U/mL penicillin, 30 ^g/mL amikacin sulfate, 100 ^g/mL streptomycin sulfate).

2. Every 7 d, the cells are dislodged from the flask using minimal contact with EDTA-trypsin reagent.

3. The suspended cells are collected via centrifugation at 1000g for 10 min and resuspended in growth medium.

4. The cell suspension is counted using a hemocytometer and diluted to allow for an inoculum of approx 105 cells/25 cm2 flask.

5. Eight milliliters of growth medium is then added to each 25-cm2 flask.

6. Within several days, the BGM cells attach to the bottom surface of the flask and form a continuous monolayer of cells.

7. To assay an environmental sample for viruses, cells are used between d 3 and 6 of their growth stage (see Note 2). The growth media is poured off and the cell monolayer rinsed with serum-less media prior to being overlaid with sample concentrate.

8. The sample inoculum volume is calculated at <0.04 mL/cm2 of monolayer surface area.

9. The test vessels are gently rocked to distribute the sample evenly and incubated at 37°C for 80-120 min.

10. The monolayer with the sample inoculum is then covered with warmed (37°C) maintenance medium (MEM/L-15 with 2% serum) and incubated at 37°C for the designated length of time (see Note 3). Positive (20 PFU/inoculum) and negative (rinse medium) controls should be assayed with environmental samples for quality control purposes.

Cytopathogenicity assays are based on the appearance of characteristic morphological changes in the cell monolayer. These visible changes are known as cytopatho-genic effects (CPE). In conventional cell culture, the cells are monitored daily for CPE, noted microscopically over time by the rounding and detachment of the cells from the surface monolayer, initially as plaques, and often followed by total destruction. CPE may be visible in >2-14 d, depending on a variety of factors including the virus type and the initial virus concentration in the inoculum (see Note 4).

3.2. Integrated Cell Culture/PCR

1. Using ICC/PCR, environmental samples are incubated in cell culture for a minimum of 5 h (see Note 3 for description of incubation conditions).

2. Freezing the cells at -70°C stops virus replication.

3. The cells are then thawed at room temperature, or 37°C, with gentle shaking to allow the ice crystals to scrape the cell monolayer from the culture flask.

4. The cell lysate, resulting from freezing and thawing, is centrifuged at 3000g to remove the cellular debris.

5. The supernatant is collected and a 10-^L aliquot is frozen for PCR amplification (see Note 5).

3.3. RT-PCR Amplification

PCR, used in combination with a culture assay, eliminates the problems of small reaction volumes, inhibitory compounds, and detection of noninfectious sequences, normally associated with molecular techniques. PCR provides a rapid, sensitive, and specific method for the detection of DNA (adenoviruses) or RNA (enteroviruses) viruses (see Note 6 for DNA virus detection protocol and PCR kit information).

Direct PCR amplification of RNA viruses is known as reverse transcriptase-PCR (RT-PCR). RT-PCR involves: (1) heat extraction of the viral nucleic acid genome; (2) transcription of the RNA to cDNA; (3) amplification of the cDNA to yield multiple copies of the target genome using DNA polymerases; and (4) confirmational analysis of the cDNA using additional amplification cycles and primer sequences internal to the original target genome (seminested PCR). A confirmational step is necessary since PCR primers may nonspecifically amplify a nontarget organism, producing a false-positive result (see Note 7).

For single PCR reactions, two primers are designed to amplify a specific region (see Note 8). One primer is sense (of the same sequence) and one antisense (having a complementary sequence) to the original target RNA genome. For seminested PCR, one of the original primers is used with a third primer, designed internal to, (nested within) the single PCR product genome. If the sense primer that is used in the original PCR is used again for seminested PCR, the internal primer must be designed in an antisense orientation or vice versa. The seminested product, therefore will be smaller than the single PCR product and will serve as a further confirmation that the correct target sequence was indeed amplified. Positive and negative controls should always be used to validate PCR primers and reaction conditions (see Note 9).

1. Heat extraction of RNA: 3.0 |iL of 10X buffer II, 7.0 |iL of 25 mMMgCl2, and 8.0 |iL of 10 mM dNTP to total 18.0 |iL.

a. If processing multiple reactions, make a master mix by combining all the above reagents.

b. Dispense 18.0 |iL into each microfuge tube.

c. Overlay reaction volume with 3 drops of mineral oil.

d. Add 10.0 |iL of environmental sample concentrate under mineral oil.

e. To extract viral RNA, heat mixture at 95°C for 5 min.

2. Reverse transcriptase reaction: 1.0 |iL of random hexamers, 1.0 |iL of AMV reverse transcriptase, and 1.0 |iL of RNase inhibitor to total 3.0 |iL.

a. Make a master mix of the above reagents, and add 3.0 |iL per reaction tube.

b. Using an automated thermal cycler, complete one cycle at 24°C, 10 min; 44°C, 50 min; 99°C, 5 min; 5°C, 5 min.

3. cDNA amplification: 7.0 |iL of 10X Buffer II, 3.0 |iL of 25 mM MgCl2 0.5 |iL of Upstream primer (50 pmol/reaction), 0.5 |iL of Downstream primer (50 pmol/reaction), 57.5 |iL of distilled water, and 0.5 |iL of AmpliTaq enzyme (2.5 U/reaction) to total 69.0 |iL.

a. Make a master mix of the above reagents, and add 69.0 |iL per reaction tube.

b. Complete one cycle at 94°C, 1 min; 50°C, 45 s; 72°C, 1 min.

c. Link to 30 cycles at 94°C, 1 min; 55°C, 45 s; 72°C, 1 min.

d. Link to one cycle at 72°C, 7 min. Store at 4°C.

3.4. PCR Confirmation

Seminested PCR is performed with an internal primer to increase the specificity while simultaneously increasing the sensitivity of the reaction. PCR results are evaluated based on the visual presence or absence of amplified product bands using gel electrophoresis.

1. Seminested PCR amplification: 10.0 ^L of 10X buffer II, 10.0 ^L of 25 mMMgCl2, 8.0 ^L of 10 mM DNTP, 0.5 ^L Upstream primer (50 pmol/reaction), 0.5 ^L of downstream primer

(50 pmol/reaction), 60.5 ^L of distilled water, and 0.5 ^L AmpliTaq Gold enzyme (2.5 U/ reaction) to total 90.0 ^L.

a. Make a master mix of the above reagents. Dispense 90.0 ^L into a new microfuge tube.

b. Overlay with 3 drops of mineral oil.

c. Add 10.0 ^L of sample from the original RT-PCR under the mineral oil.

d. Complete 20-25 cycles at 94°C, 1 min; 55°C, 45 s; 72°C, 1 min.

e. Link to one cycle at 72°C, 7 min. Store at 4°C.

All the above protocols require about 7 h to complete using a Gene Amp DNA thermal cycler 480 or 4 h with a Gene Amp 9600 series thermal cycler (see Note 10). Reagents may be adjusted in equal proportions for smaller volume reactions. 2. Gel electrophoresis. Amplicons of PCR are visualized by horizontal agarose gel electrophoresis using a 3% intermediate melting temperature agarose gel (Metaphor agarose; FMC Bioproducts).

a. Combine 15.0 ^L of PCR product with 3.0 ^L Ficoll loading buffer (20% Ficoll, 1% sodium dodecyl sulfate, 0.25% bromphenol blue, and 0.1 M disodium EDTA, pH 8.0).

b. Load samples into wells of premade gel slab submerged in 1% tris-acetate/EDTA (TAE) buffer.

c. Subject to electrophoresis at 100 V for 2-3 h with cooling recirculation.

d. Remove gel and submerge in an ethidium bromide DNA staining solution for 15 min.

e. Resubmerge gel in distilled water for 30 min to remove background stain.

f. Visualized stained amplicons by exposure to a 302-nm UV-transilluminator.

4. Notes

1. The ICC/PCR method can be easily adapted to other environmental viruses. Additional cell lines include: (1) Hep-2 from human epidermoid carcinoma cells for the growth of human adenoviruses; (2) PLC/PRF/5 cells from a human primary hepatocellular carcinoma for the isolation of human enteric adenovirus types 40 and 41; and (3) FRhK cells from fetal rhesus monkey kidneys for the growth of HAV, and (d) CaCO2 cells from a human colorectal adenocarcinoma for the growth of multiple enteric viruses. Since the development of ICC/PCR (1), a variety of similar protocols have been developed for the detection of additional human viruses (2,6,9,10). A variety of cell lines can be purchased from the American Type Culture Collection (Manassas, VA).

2. To prevent potential contamination of cell lines, cell growth and sample inoculation should be performed in separate rooms with designated equipment. Some viruses, such as noncytopathogenic strains, may grow unnoticed with the cell line but may interfere with subsequent detection methods.

3. Positive ICC/PCR detection is a function of: (1) initial virus inoculum; (2) incubation time; and (3) the number of replicate flasks (equivalent volume) examined. Higher concentrations (>10 PFU/flask) of poliovirus strain LSc-2ab, a cytopathogenic virus, may be detected in seven of eight replicate flasks after only 5 h of incubation. For 100% positive detection, at a poliovirus concentration of >10 PFU/flask, a minimum of 10 h is required. For the target concentration of 1 PFU/flask inoculum, >20 h is recommended. Similarly, higher levels (100 PFU/flask) of HAV strain HM175, a noncytopathogenic virus, may be detected as soon as 12 h post incubation, but this fastidious virus requires a minimum of two to three replicate flasks to evaluate its presence or absence effectively. Infectious HAV levels of 10 and 1 PFU/flask are detectable after 48 and 72 h and require two to three replicate flasks, respectively. Therefore, for a given virus and a desired detection limit, one must evaluate the minimum incubation time and equivalent volume required to ensure reliable results using ICC/PCR.

4. The ICC/PCR method is most effective when cell cultures are stopped prior to complete lysis of the cell monolayer. Over time, PCR-inhibitory substances build up in the culture flasks, requiring sample purification. If inhibition does occur, reduce the culture assay time or use a resin column purification technique (11,12) as follows:

a. Test samples for inhibition by seeding low levels of target virus into the PCR (1-10 PFU).

b. If inhibition is evident, apply 50.0 ^L of sample to a layered Sephadex G-25 coarse/ Chelex-100 resin column in a Silane-treated, glass wool plugged, 1-mL syringe (approx 0.5 mL vol of each resin are used with a wet particle size range of 87-510 and 150-300 ^m, respectively).

c. Allow sample to adsorb for 10 min.

d. Centrifuge column at 3000g, and collect samples for RT-PCR analysis.

e. Retest for inhibition as in step a.

f. If inhibition remains, pass the sample through a second layered column or through one with greater size exclusion capacity, such as Sephadex G-200/Chelex-100 (G-200 wet particle size range of 30-380 ^m).

5. A negative ICC/PCR result at incubation time zero is an assurance of viable virus detection. For the time zero replicates, samples are placed on the cell monolayer and incubated, as described in Note 3, to allow virus attachment to the cells. After the cells are covered with maintenance media, the assay is stopped, prior to production of progeny virus. Therefore, if all replicate flasks are negative at time zero and positive at time one, virus growth was necessary for positive integrated CC/PCR results, indicating the detection of viable viruses only.

6. Amplification protocols for a DNA virus are identical to the reaction conditions described above for cDNA targets. PCR kits offer time-saving advances, providing all reagents needed for complete reactions, ensuring high-quality control standards, and offering of single-tube RT-PCR amplification kits and premeasured PCR optimization kits (Promega; Roche Molecular Systems).

7. Cytotoxic effects, not caused by viruses but rather sample toxins, typically inactivate the cell monolayer within 1-3 d of the assay. If the target virus is fastidious, and growth does not occur within this time, cytotoxic effects may be reduced by: (1) changing the maintenance medium at the first sign of cytotoxicity; (2) diluting the sample inoculum with distilled water or cell culture media (1:2 or 1:4 dilution); (3) reducing the sample adsorption time to 15 or 30 min; (4) after the initial sample adsorption with the cells, thoroughly rinsing the sample inoculum from the cell monolayer with rinse media; or (5) pretreating the cells with a solution of 8.5 g NaCl in a final volume of 980 mL of reagent-grade water, autoclaved and cooled to room temperature. Add 20 mL of serum and mix thoroughly. Add 0.25 mL/cm2 of cell surface area. This technique reduces the virus titer and is used only when necessary.

8. Primer design is also very important to ensure a reliable PCR result. In general, primers should have the following characteristics:

a. Primers are typically between 17 and 30 bp long.

b. Ideally, the amplification product should be between 100 and 500 bp (although possible, amplification of longer products is more difficult to optimize).

c. Primers must not be complementary to one another and should uniquely amplify the intended target.

d. The GC content should be >40% to enable higher annealing temperatures.

e. Internal secondary structures within the primers should be avoided.

9. Laboratories using PCR on a routine basis must develop quality control procedures to prevent false positive results and maintain the integrity of the PCR (13). False-positive results owing to contamination can be reduced by the following precautions:

a. Establish pre- and post-PCR work stations. These areas should be in separate rooms or as far away from one another as possible. By doing this, the PCR-amplified product is separated from prereaction preparations.

b. If possible, dedicate sets of unique supplies and pipeting devices for each set of primers.

c. Autoclave buffer solutions and use HPLC-grade water. (Primers, dNTPs, and enzymes cannot be autoclaved.)

d. Aliquot reagents to minimize the number of repeated samplings from a single stock.

e. Use disposable gloves and change frequently, taking care not to cross-contaminate pre- and post-PCR stations.

f. Spin down tubes prior to opening to reduce aerosolization of the sample.

g. Use positive displacement pipets or aerosol-resistant tips to avoid contamination via aerosols.

h. Premix reagents before dividing into aliquots, i.e., use master mixes.

i. Add all reagents before sample DNA, capping each tube before opening the next.

j. Use hot start methodologies, or heat-activated enzymes (i.e., AmpliTaq Gold; Roche Molecular Systems) to reduce preamplification mispriming. (Hot start is a procedure whereby the PCR is first set up at 80°C without one critical component, the Taq polymerase, heated to 94°C to denature the template, and then cooled to around 80°C. The polymerase is then added after the chance for mispriming is reduced. Heat-activated enzymes cause a similar effect and do not start performing until the higher temperatures are reached for a certain period). k. Use the minimum number of PCR cycles possible for a given sample. l. Contamination can be checked by the use of negative controls and should be run at the beginning and end of the set of PCR tubes as a minimum requirement. Negative controls should be made from the same master mix as the other sample reactions but contain no added RNA or DNA template. Standard protocols may need to be optimized for various types of samples. The number of amplification cycles can range from 15 to 30; primer annealing temperatures typically range from 35°C to 60°C. Mg2+ is required in the reaction, but the optimal concentration range is from 1.5 to 4 mM.

10. Reaction times may be decreased by as much as one-half using the thin-walled PCR tubes and an accommodating thermal cycler (i.e., Gene Amp 9600 PCR thermal cycler, Perkin Elmer, Roche Molecular Systems). This equipment eliminates the need for mineral oil in the reaction and allows for more rapid heat transfer in the tubes and thus shorter reaction times. Furthermore, real-time PCR assays offer a more rapid completion of PCR amplification of samples, with an internal detection system using double-stranded DNA-specific dyes or internal probes. As the PCR product accumulates, the product is detected simultaneously, eliminating the need for gel electrophoresis. Although most efficient, real-time PCR equipment is very costly (upwards of $50,000).


1 Reynolds, K. A., Gerba, C. P., and Pepper, I. L., (1996) Detection of infectious enteroviruses by an integrated cell culture-PCR procedure. Appl. Environ. Microbiol. 62, 1424-1427.

2 Reynolds, K. A., Gerba, C. P., Abbaszadegan, M., and Pepper, I. L., (2001). ICC/PCR detection of enteroviruses and hepatitis A virus in environmental samples. Can. J. Microbiol. 47, 153-157.

3 Reynolds, K. A., Gerba, C. P., and Pepper, I. L., (1997) Rapid PCR-based monitoring of infectious enteroviruses in drinking water. Wat. Sci. Tech. 35, 423-427.

4 Blackmer, F., Reynolds, K. A., Gerba, C. P., and Pepper, I. L., (2000) Use of integrated cell culture-PCR to evaluate the effectiveness of poliovirus inactivation by chlorine. Appl. Environ. Microbiol. 66, 2267-2268.

5 Abbaszadegan, M., Huber, M. S., Gerba, C. P. and Pepper, I. L. (1993) Detection of enteroviruses in groundwater with the polymerase chain reaction. Appl. Environ. Microbiol. 59, 1318-1324.

6. Hurst, C. J. and Reynolds, K. A., (2002) Detection of viruses in environmental waters, sewage and sewage sludges. In: Manual of Environmental Microbiology (Hurst, C. J., ed.). ASM Press, Washington, DC, pp. 244-253.

7. Hurst, C. J. and Reynolds, K. A., (2002) Sampling viruses from soil. In: Manual of Environmental Microbiology (Hurst, C. J., ed.). ASM Press, Washington, DC, pp. 527-534.

8. United States Environmental Protection Agency, (1995) Virus Monitoring Protocol for the Information Collection Requirements Rule. Document number: EPA/814-B-95-002. June 1995. USEPA, Cincinnati, OH, pp. 1-70.

9 Chapron, C. D., Ballester, N. A., Fontaine, J. H., Frades, C. N., and Margolin, A. B., (2000) Detection of astroviruses, enteroviruses, and adenovirus types 40 and 41 in surface waters collected and evaluated by the information collection rule and an integrated cell culture-nested PCR procedure. Appl. Environ. Microbiol. 66, 2520-2525.

10. Greening, G. E., Hewitt, J., and Lewis, G. D. (2002) Evaluation of integrated cell culture-PCR (C-PCR) for virological analysis of environmental samples. J. Appl. Microbiol. 93, 745-750.

11. Straub T. M., Pepper, I. L. and Gerba, C. P. (1994) Detection of naturally occurring enterovi-ruses and hepatitis A virus in undigested and anaerobically digested sludge using the poly-merase chain reaction. Can. J. Microbiol. 40, 884-888.

12 Reynolds, K. A., Roll, K., Fujioka, R. S., Gerba, C. P., and Pepper, I. L., (1998) Incidence of enteroviruses in Mamala Bay, Hawaii using cell culture and direct polymerase chain reaction methodologies. Can. J. Microbiol. 44, 598-604.

13. Pepper, I. L. and Pillai, S. D., (1994) Detection of specific DNA sequences in environmental samples via polymerase chain reaction. In: Methods of Soil Analysis, Part 2. Microbiological and Biochemical Properties. ASA, CSSA, SSSA, Madison, WI, pp. 707-726.

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