## Quantification o f Enterovirus RNA i n Sludge Samples Usin g Single Tube Real-Tim e RT-PC R BioTechniques 29:88-93 (July 2000)

ABSTRAC T We have developed a quantitative RT

PCR method that can be used to determin e the amount of enterovirus RNA in urba n sludge samples. This method combines Ta q Ma n Ò technology with the ABI Pris m Ô 7700 real-time sequence detection system . We optimized a one-step RT-PCR that use s a dual-labeled fluorogenic probe to quantif y the 5 ¢ noncoding region of enteroviruses . F or accurate quantification of the numbe r of copies, a Mahoney type 1 poliovirus RN A standard was designed and produced usin g genetic engineering. This fragment, quant i
fied using the Ribogree n Ò method, was use d in serial dilutions as an external standard. The method had a 7-log dynamic range ( 5 to 2  ́ 1 0 7). PCR inhibitors were remove d by extracting viral RNA (after virus conce n tration) using the RNeasy Ò mini kit with added polyvinylpyrrolidone (PVP) and ru n ning the amplification reaction with a mi x ture containing PVP and T4 gene 32 pro tein. This real-time quantification o f enterovirus RNA allows large numbers o f samples to be screened. Its sensitivity, si m plicity and reproducibility render it suitabl e as a screening method with which to cha r acterize enteroviruses, the presence of i n fectious particles being subsequently co n firmed by cell culture . INTRODUCTIO N There are generally two kinds o f methods for detecting enteric viruses i n environmental samples, depending o n whether they are based on cell cultur e infectivity or on molecular detectio n methods such as PCR followed by n u cleic acid hybridization (16). Compare d to time-consuming cell culture assays , PCR is rapid, sensitive and specific. It i s also capable of detecting non-cytopath

ogenic, slow-growing and rare viruse s in complex environmental samples suc h as sewage or sludge (20). Current am plification techniques are either qualit a tive (10) or quantitative with the me a surement of the amount of produc t amplified as their end point (25). In this study, we suggest a single

tube, one-step, real-time R T -PCR assay for the quantification of enteroviruse s in sludge samples using TaqMan  technology (Roche Molecular Bi o chemicals, Meylan, France). This tec h

nology makes use of the 5 ′→ 3 ′ nucl e olytic activity of AmpliTa q  DNA polymerase and uses primer pairs i n conventional amplification and a dual

labeled fluorogenic probe (5,13). Thre e major difficulties need to be overcom e in the development of such a technique : ( i ) because enteroviruses are RN A viruses, RT

-PCR needs to be optimize d in a single step to keep manipulatio n time to a minimum; ( i i ) because treate d sludge contains only small quantities o f enterovirus, a highly sensitive tec h

nique is required; and ( ii i ) the pures t possible genetic material needs to b e extracted because sludge samples co n tain numerous organic compound s (polyphenols and humic acids) an d heavy metals that are liable to inhibi t amplification enzymes (7,17,21). The use of polyvinylpyrrolidon e (PVP) in genome extraction and the T 4 gene 32 protein during amplificatio n enabled us to achieve a complete prot o col, from the extraction of virus fro m sludge to the counting of genom e copies. Here, we present the first result s obtained with samples of natural an d artificially contaminated sludge. W e compare these results with measur e ments made using cell culture, whic h remains the reference technique . MATERIALS AND METHOD S Residual Sludg e Three types of sludge were sample d from two waste-water treatment plant s in Lorraine, France. Primary sludg e was taken from the primary decantin g ponds of the Nancy and Metz treatmen t plants; activated sludge and thickene d sludge from a secondary decantin g pond were taken from the Metz plant . Samples of thickened sludge (78% dry solids) loaded with 7 × 1 0 4 and 7 × 1 0 3 pfu poliovirus 1 vaccine (Sabin) wer e used as controls . Virus Extraction and Concentratio n T echniqu e Virus was eluted from sludge sam ples using the technique described b y Ahmed and Sorensen (1). A volume o f sludge providing 10 g (dry solids), t o which 100 mL 10% beef extract (p H 9.0) (Oxoid, Basingstoke, UK) ha d been added, was stirred at 500 rpm fo r 30 min. The mixture was sonicated o n ice (100 W, 0.9 s) for 5 min in 1-mi n steps, mixed again for 5 min and the n centrifuged at 500 0 × g for 1 h at 4 ° C . The extract consisted of the supernatant , neutralized to pH 7.2. This extract wa s concentrated using polyethylene glyco l (PEG) 6000 (Prolabo, Fontenay sou s bois, France) and precipitated accordin g to the Lewis and Metcalf techniqu e (12). An 8% final concentration (w/v ) PEG 6000 (in PBS, pH 7.2, bi o Merieux, Lyon, France) was added t o the extract. After vigorous agitation, th e mixture was stored overnight at 4°C an d then centrifuged at 1 0 00 0× g for 90 mi n at 4°C. The pellet was resuspended i n 10 mL PBS. Finally, this concentrat e was decontaminated by adding a one

third volume of chloroform (10-mi n centrifugation at 150 0 × g ) followed b y 200 μL antibiotic cocktail (penicilli n 1 0 0 000 IU/mL, colimycin 2 5 0 000 IU/ mL, streptomycin 100 mg/mL and am photericin B 0.5 mg/mL) . Quantification of Infectiou s Enterovirus Using Cell Cultur e Quantities of the present infectiou s enterovirus were measured by inoc u lating the decontaminated conce n trates in vitro into buffalo gree n monkey cell cultures in 96-well m i

croplates. All cultures were inoculate d in duplicate using 40 wells for each d i

lution (fivefold serial dilutions). Eac h well was filled with 50 μL inoculu m and 200

- μL nutritive medium [Eagl e minimum essential medium (Eurobio , Les Ulis, France), to which 5% ne w born calf serum had been added] co n taining 1.5 × 1 0 5 cells/mL. The cu l

tures were incubated at 3 7 ° C in 5 % C O 2 for five days. Virus density wa s 

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8 8 BioTechnique s Vol. 29, No. 1 (2000 )determined from the cytopathic effect s

observed after duplicate inoculation o f cell layers with three successive dil u tions of the sample. The cytopathic e f

fect was confirmed by two serial pa s sages. The mean viral concentratio n for each sample was estimated usin g the most probable number (MPN ) method with software devised by Mau l (15). Results were expressed in th e most probable number of colony uni t (MPNCU)/mL sample and then co n verted to MPNCU/10 g sludge (dr y solids) to take into account any vari a tions in sludge dryness. Extraction of Viral RN A Enterovirus RNA was extracte d from 250 μL concentrate using th e RNeas y  plant mini kit (Qiagen , Courtaboeuf, France) according to th e manufacturer’s instructions, with th e following exception: the kit RLT buffer was modified by the addition of 2 % (w/v) PVP 4 0 000 (Sigma, St. Quentin , France) (4,22,26) . Fluorogenic RT-PC R To design the primers and probe fo r the TaqMan technology, the mos t c o nstant genome region in enteroviru s es, the 5 ′ -noncoding region ( 5 ′ NCR) , was chosen. The software Primer E x press was used and identified primer Ev1 [ 5 ′ -G ATTGTCACC AT AAGC A

GC- 3 ′ ] (position 579–597), primer Ev 2 [ 5 ′ -CCCCTGAATGCGGCTAATC- 3 ′ ] (position 451–469) and Ev-probe [ 5 ′

- FAM-CGGAAC C GACTACTTTGG G

TGTCCGT-TAMRA-Phosphor- 3 ′ ] (p o sition 532–557). These primers were tested in the GenBank database an d fou n d to be pan-specific and to cove r all enteroviruses present in GenBank . The reaction mixture (final volum e 25 μL) was prepared in a single tube a s follows: 1 × TaqMan buffer A, 5.5 m M MgC l 2 (both from PE Biosystems , Courtaboeuf, France); 500 μM dNTP s (Roche Molecular Biochemicals); 50 0 nM reverse primer Ev1, 400 nM primer Ev2 (both from Genosys, Pampisford , UK); 120 nM Ev-probe (Eurogentec , Serraing, Belgium); 6% glycerol (Pr o labo); 1.7% PVP-25 (Serva, Paris , France); 1.5 μ g T4 gene 32 protei n (Amersham Pharmacia Biotech, Orsay , France); 5 UI Mu LV and 2.5 UI Ampli

T a q  Gold (both from PE Biosystems ) and 10 UI RNasi n  (Promega, Charbo n nière, France). Twenty microliters of th e reaction mixture were added to the PC R tubes containing 5 μL RNA from one o f the sludge samples or RNA from th e constructed standard in serial dilution . Enterovirus RNA was reverse tran scribed into cDNA (45 min at 50°C) an d the 147-bp fragment was amplified b y PCR (45 cycles of 9 4 ° C for 15 s an d 6 0 ° C for 1 min) on an ABI Prism 7700 . Enterovirus RNA Standard fo r Absolute Quantificatio n An RNA standard representing th e 5’NCR of enterovirus RNA was synth e sized in vitro as follows: cDNA fro m

V ol. 29, No. 1 (2000 ) BioTechniques 8 9Mahone y typ e 1 polioviru s wa s amplifie d b y PC R usin g primer Ev1Clo n 5 ′ -TGG C

CAATCGAATTCGCT TTA- 3 ′ (positio n 612–632 ) an d prime r Ev2Clo n 5 ′ -CT A

CATAAGG

A TCCTCCGGCC- 3 ′ (pos i

tion 432–452) modified by the introdu c tion of restriction sites ( Ba m HI an d Ec o RI). The 201-bp fragment was ide n tified on a 1.5% agarose gel, excised , isolated from the gel and ligated into th e polylinker of a pT7-blue vector (Nov a gen, Madison, WI, USA). The recomb i

nant plasmid (pT7-EV) was transforme d into E. col i (Novagen) and a positiv e colony was amplified in LB mediu m containing tetracycline and ampicillin . Plasmids were purified by alkaline lysi s (2), linearized, purified by gel ele c trophoresis and extracted from the ge l using a plasmid maxi kit (Qiagen). Standard plasmids were quantifie d using spectrophotometric analysis. Th e sequence and orientation were verifie d by automatic sequencing. Linearize d plasmid (1.5 μ g) was used to perform an in vitro transcription reaction with T7 DNA polymerase (Roche Molecula r Biochemicals) at 37°C for 2 h. After di

gestion with RNase-free DNase, the re sulting RNA transcripts were purifie d with phenolchloroform and precipitate d with isopropanol. The RNA transcript s were dissolved in sterile, RNase-fre e water and quantified by spectrofluor o metric analysis using Ribogree n  (I n terchim, Montluçon, France). The tite r was 1 0 1 0 copies /μL. Dilutions (fiv e copies to 2 × 1 0 7 ) of the RNA standard were made in TE buffer containin g RNasin. Aliquots of the diluted RN A stock were frozen immediately at

- 7 0 ° C. Each aliquot was used once onl y for a fluorogenic RT

-PCR . Analysis of Fluorescence Signals o n the ABI Prism 770 0 Real-time fluorescence measure ments were taken and the threshold c y cle ( C t ) value for each sample was calc u lated by determining the point at whic h fluorescence exceeded a threshold limi t (10 times the baseline S D ). A standard graph of the C t values obtained from th e serially diluted external RNA standard was compiled. The correlation coeff i

cient and the slope were calculated. C t values from the sludge samples were plotted on the standard curve, and th e number of copies was calculated aut o matically by Sequence Detector v1.6 . Samples were defined as negative i f their C t values exceeded 45 cycles . RESULTS AND DISCUSSIO N During the development of thi s method, the optimal conditions for R T

PCR were determined by amplifying a range of the standard RNA. A proble m we encountered was that of sensitivity (Figure 1A; detection limit = 1.6 × 1 0 4 copies) related to the secondary stru c tures present in the 5 ′ NCR of the e n terovirus genome. For this reason, w e decreased the RT

/ T a q ratio and used th e T4 gene 32 protein, usually considere d to be a ssDNA binding protein and a dsDNA helicase (11,3). Figure 1 B demonstrates the benefits of adding thi s protein to the reaction mixture (in quan tities of 1.5 μ g/reaction) on the amplif i

cation yield of the standard RNA . The detection limit, intra-assay an d 

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9 0 BioTechnique s Vol. 29, No. 1 (2000 ) Figure 1. Comparison of intensity and amplification quality in sludge extracts . These extracts co n tains seven successive fivefold dilutions of standard RNA (25 to 4 × 1 0 5 copies) in the presence and ab

sence of T4 gene 32 protein (Fam C3-D3 = 25, Fam C4-D4 = 125, Fam C5-D5 = 640, Fam C6-D6 = 3. 2 × 1 0 3 , Fam C7-D7 = 1.6 × 1 0 4 , Fam C8-D8 = 8 × 1 0 4 , Fam C9-D9 = 4 × 1 0 5 ). 1A: Amplification in th e absence of T4 gene 32 protein, three dilutions are amplified. 1B: Amplification in the presence of T 4 gene 32 protein, all dilutions are amplified . A

Binterassay variability were determined i n

10 replicates of tenfold dilution stan dards, from 5 to 5 × 1 0 6 RNA copies , amplified in eight independent assays . Intra-assay C V s varied between 6.34 % and 0.74%, and interassay C V s betwee n 4.51% and 0.66%. The values of 6.34 % and 4.51% obtained with low RNA co n centrations agree with those found i n other TaqMan reactions (3.4% an d 6.2%) (6,14). A variability of five copie s of the standard is acceptable, and no ne g ative result was obtained. The sensitivit y of the RT-PCR is therefore five copies . Other problems encountered wer e those caused by the complexity of th e material, such as the residual sludge . Sludges contain numerous inorgani c and organic compounds—humic acid s and polyphenols, for example—whic h are liable to form complexes with n u cleic acids and inhibit amplification e n zymes. Figure 2 shows the presence o f such inhibitor substances on the graph s fam-D1 produced by the Sequence D e tector software when 8 × 1 0 4 copies o f enterovirus RNA from a sludge sampl e were amplified. PVP is described in th e literature as forming complexes with phenolic compounds (9,26). For max i

mum effect, it was added both at th e RNA extraction stage and to the PC R mixture (Figure 2, Fam-D6). Th e amount of PVP required in the RN A extraction process was fixed at 2% as i n the cetyltrimethylammonium bromid e (C TAB) extraction protocol (22), whil e the amount required in the amplific a tion mixture was determined by addin g various quantities between 1% and 2% , as did Koonjul et al. (9). The optimu m PVP amount was found to be 1.7% . The analytical sensitivity of ou r method, considering all extractio n steps, is 2000 copies/10 g (dry solids ) of sludge. However, lower numbers can be found because the linearity of th e standard curve permits the interpolatio n of values below the lower limit of th e dynamic range (14). Therefore, the th e oretical detection limit of our RT-PC R can be adjusted to 400 copies/10 g (dry solids). This sensitivity is compatibl e with requirements accepted in the lite rV ol. 29, No. 1 (2000 ) BioTechniques 9 1 Results from RT-PC R Results from cell cultur e Sample s (number of copies/10 g dry solids ) (MPNCU/10 g dry solids ) control 1 (7 × 1 0 4 pfu ) 1.5 × 1 0 6 4.9 × 1 0 4 control 2 (7 × 1 0 4 pfu ) 1.4 × 1 0 6 3.5 × 1 0 4 control 3 (7 × 1 0 4 pfu ) 8 × 1 0 6 3.4 × 1 0 4 control 4 (7 × 1 0 4 pfu ) 1 × 1 0 6 1.4 × 1 0 4 control 5 (7 × 1 0 4 pfu ) 7 × 1 0 6 2.2 × 1 0 4 control 6 (7 × 1 0 3 pfu ) 2 × 1 0 5 1.6 × 1 0 3 control 7 (7 × 1 0 3 pfu ) 3 × 1 0 5 3.6 × 1 0 3 control 8 (7 × 1 0 3 pfu ) 1 × 1 0 5 1.7 × 1 0 3 1 P 5 4 72 0 1 0 2 P 2 3 0 40 0 224 2 3 P 1 1 5 20 0 32 6 4 A 566 4 10 7 5 A 1 4 40 0 27 8 6 A <40 0 < 3 7 T 1 4 40 0 < 3 8 T 1 4 40 0 7 2 9 T < 40 0 < 3 Measurements use R T

-PCR and cell culture in eight sludge samples artificiall y contaminated with 7 × 1 0 4 and 7 × 1 0 3 pfu poliovirus 1 and nine natural sludg e samples of three different types (P = primary, A = activated, T= thickened)
. Table 1: Enterovirus Quantification Measurements.ature as suitable for the detection of e n teroviruses in sludge from waste-wate r treatment plants (10,20,23,24) . This fluorogenic RT-PCR protoco l was first evaluated on eight samples o f artificially contaminated thickene d sludge and then applied to nine natural sludge samples. The results, presente d in Table 1, were compared to measure ments made of virus quantities b y means of cell culture. For the sample s artificially contaminated with 7 × 1 0 4 or 7 × 1 0 3 pfu, the mean values as me a sured by RT-PCR and cell culture, r e spectively were 3.78 × 1 0 6 copies/10 g and 3.08 × 1 0 4 MPNCU/10 g (n = 5) o r 2 × 1 0 5 copies/10 g and 2.96 × 1 0 3 M P

NCU/10 g (n = 3) (Table 1). In the nat ural sludge samples, two samples wer e negative (< 400 copies/10 g) accordin g to RT -PCR and three according to cel l culture (< 3 MPNCU). The quantity of genomic RNA me a sured was always larger than the nu m ber of infectious particles, being on av erage 150 times greater for natural sludge and 120 times greater for cont a minated sludge. This can be explaine d by ( i ) the aggregation of infectious pa rticles into clumps that, while consistin g of several viruses, only cause one are a of lysis in a cell culture; ( i i ) the variabl e effects of cell culture inhibitory su b

stances that might still be present in th e concentrates; and ( ii i ) the ability of RT

PCR to detect inactivated viruses. Thes e results, which agree with those reporte d in the literature (8,18,19), show a shar p decrease in quantities of virus presen t from primary sludge through activate d and thickened sludges . The fluorogenic RT

-PCR techniqu e developed in this study allows real-time analysis of the amplification reactio n and calculation of genomic RNA quan tities at the start of the exponential phas e of amplification. The fluorescence si g nal caused by cleavage of the TaqMan fluorogenic probe can only be generate d if the target sequence has been ampl i

fied, which gives the reaction a high le v el of specificity. Our protocol is sens i

tive, reproducible and rapidly execute d on multiple samples. These feature s make it an excellent large-scale scree n ing tool for sludge from waste-wate r treatment plants and for monitorin g sewage processing procedures, eve n though the presence of infectious parti

cles must be confirmed by cell culture , as current regulations require . REFERENCE S 1 . Ahmed, A.U. and D.L. Sorensen . 1995. Kine t ics of pathogen destruction during storage o f dewatered biosolids. Wat. Environ. Res . 6 7 :143-50 . 2 . Birnboim, H.C. and J. Doly . 1979. A rapid al

kaline extraction procedure for screening r e combinant plasmid DNA. Nucleic Acids Res . 7 :1513-1523 . 3 . Chandler, D.P., C.A. Wagnon and H. Bolton , Jr . 1998. Reverse transcriptase (RT) inhibitio n of PCR at low concentrations of template an d its implications for quantitative RT- PCR. Appl . Environ. Microbiol. 6

4 :669-677 . 4 . Felske, A., B. Engelen, U. Nübel, and H. Bac k haus . 1996. Direct ribosome isolation from soi l to extract bacterial rRNA for community anal y sis. Appl. Environ. Microbiol. 6 2 :4162-4167 . 5 . Gibson, U.E., C.A. Heid and P.M. Williams . 1996. A novel method for real time quantitativ e RT-PCR. Genome Res. 6 :995-1001 . 6 . Gut, M., C.M. Leutenegger, J.B. Huder, N.C . Pedersen and H. Lutz . 1999. One-tube fluor o genic reverse transcription-polymerase chai n reaction for the quantitation of feline coro n aviruses. J. Virol. Methods 7 7 :37-46 . 7 . Higuchi, R . 1992. Simple and rapid preparatio n of samples for PCR, p. 31-38. I n H.A. Erlic h (Ed.), PCR Technology. W.H. Freeman an d Co., New York . 8 . Hu, C.J., R.A. Gibbs, G.E. Ho, P. Phillips an d I. Unkovich . 1995. Pathogen densities in wast e water sludge treated by anaerobic digestion an d dewatering. 3rd International Conference A p propriate Waste Management Technologies fo r Developing Countries . 9 . Koonjul, P.K., W.F. Brandt, J.M. Farran t and G.G. Lindsey . 1999. Inclusion of pol y vinylpyrrolidone in the polymerase chain rea c tion reverses the inhibitory effects of pol y phenolic contamination of RNA. Nucleic Acid s Res . 2 7 :915-916 . 10 . Kopecka, H., S. Dubrou, J. Prevot, J. Mar e chal and P

.J.M. Lopez . 1993. Detection o f naturally occurring enteroviruses in waters b y reverse transcription, polymerase chain rea c tion, and hybridization. Appl. Environ. Micr o biol. 59 :1213-1219 . 11 . Kreader, C.A. 1996. Relief of amplification i n hibition in PCR with bovine serum albumin o r T4 gene 32 protein. Appl. Environ. Microbiol . 6 2 :1102-1106 . 12 . Lewis, G.D. and T.G. Metcalf. 1988. Polyeth

ylene glycol precipitation for recovery of path

ogenic viruses including hepatitis A and human rotavirus from oyster, water and sediments . Appl. Environ. Microbiol. 5 4 :1983-1988 . 13 . L ivak, K.J., S.J. Flood, J. Marmaro, W . Giusti and K. Deet z . 1995. Oligonucleotide s with fluorescent dyes at opposite ends provide d a quenched probe system useful for detectin g PCR product and nucleic acid hybridization . PCR Methods Appl. 4 :357-362 . 14 . Martell, M., J. Gomez, J.I. Esteban, S. Saul e da, J. Quer, B. Cabot, R. Esteban and J . Guardia . 1999. High-throughput real-time r e verse transcription-PCR quantitation of hepati

tis C virus RNA. J. Clin. Microbiol. 3 7 :327

- 332 . 15 . Maul, A. 1991. Aspects statistiques des meth

odes de quantification en virologie, p. 143-171 . I n Lavoisier (Ed.), Virologie des milieux h y driques. Schwartzbrod Tec. & Doc., Paris . 16 . Metcalf, T.G., J.L. Melnick and M.K. Estes . 1995. Environmental virology: from detectio n of virus in sewage and water by isolation t o identification by molecular biology—a trip o f 

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9 2 BioTechnique s Vol. 29, No. 1 (2000 ) Figure 2. Graph showing fluorescence intensity as a function of numbers of amplification cycles . Co m parison of amplification intensity of the same sludge concentrate (10 μ L) containing 8 × 1 0 4 copies of RNA under different reaction conditions. Fam-D1: RNA extraction and amplification without PVP; Fam-D2: RNA extraction with PVP, amplification without PVP; Fam-D6: RNA extraction and amplification with PVP .over 50 years. Annu. Rev. Microbiol. 4 9 :461

-

487 . 17 . Muller-Wegener, U. 1988. Interaction of h u mic substances with biota, p. 179-192. I n F.H . Frimmel and R.F. Christman (Eds.), Humi c Substances and Their Role in the Environment . John Wiley & Sons, Inc., New York . 18 . Pederson, D. 1981. Density levels of pathoge n ic microorganisms in municipal waste wate r sludge: indicator organism in sonoran desert soil amended with sewage sludge. J. Environ . Sci. Health A2 8 :1287-1302 . 19 . Soares, A.C., T.M. Straub, I.L. Pepper an d C.P. Gerba . 1994. Effect of anaerobic dige s tion on the occurrence of enteroviruses and G i

ardia cysts in sewage sludge. J. Environ. Sci . Health A2 9 :1887-1897 . 20 . Sobsey, M.D. 1994. Molecular methods to d e tect viruses in environmental samples., pp. 387

- 400. I n R.C. Spencer, E. P

. Wright and S.W.B . Newsom (Eds.), Rapid Methods and Autom a tion in Microbiology and Immunology. Inte r cept Ltd., Andover, Hampshire, UK . 21 . Staub, U., H. Polivka and H.J. Gross . 1995 . Two rapid microscale procedures for isolatio n of total RNA from leaves rich in polyphenol s and polysaccharides : application for sensitiv e detection of grapevine viroids. J. Virol. Meth

ods 5 2 :209-218 . 22 . Stewart, N.C., Jr. and L.E. Vi a . 1993. A rapi d CTAB DNA isolation technique useful fo r RAPD fingerprinting and other PCR applic a tion. BioTechniques 1 4 :748-749 . 23 . Straub, T.M., I.L. Pepper and C. P . Gerba . 1994. Detection of naturally occurring e n teroviruses and hepatitis A virus in undigeste d and anaerobically digested sludge using th e polymerase chain reaction. Can. J. Microbiol . 4 0 :884-888 . 24 . S traub, T.M., I.L. Pepper, M. Abbaszadega n and C.P. Gerba . 1994. A method to detect e n teroviruses in sewage sludge-amended soil u s ing the PCR. Appl. Environ. Microbiol . 6 0 :1014-1017 . 25 . Tsai, Y.-L. and S.L. Parker . 1998. Quantific a tion of poliovirus in seawater and sewage b y competitive reverse transcriptase polymeras e chain reaction. Can. J. Microbiol. 44 :35-41 . 26 . Wan, C.-Y. and T.A. Wilkins. 1994. A mod i

fied hot borate method significantly enhance s the yield of high-quality RNA from cotton . Anal. Biochem. 22 3 :7-12 . This study is based on work supported by A.d.e.m.e (the French environment and ene rgy management agency) and Anjou Reche r che. We thank INSERM U437 and Perki n Elmer for technical support with the AB I Prism 7700. Address correspondence to D r . Serge Monpoeho, Laboratoire de Virologi e CHU Hotel Dieu, 9 quai moncousu, 4403 5 Nantes, France. e-mail: sbillaudel@chu

nantes.f r Received 23 July 1999; accepted 1 5 March 2000
. 

### S. Monpoeho, A. Dehé e 2 

### , B . Mignotte, L. Schwartzbro d 1 

### , V . Marecha l 2 

### , J.-C. Nicola s 2 

### , S. Billaudel and V

. Férré Laboratoire de Virologie CH U de Nantes, Nante s 1 

### Laboratoire de Virologi e faculte de pharmaci e Nancy, Nanc

y 2 

### Laboratoire de Virologi e hopital Rothschild Paris, Franc e