DETECTION OF ENTEROVIRUSES AND HEPATITIS A VIRUS 

IN ENVIRONMENTAL SAMPLES BY GENE PROBES AND 

POLYMERASE CHAIN REACTION 

Ricardo De Leon1 

Research Associate 

Carol Shieh2 

Research Associate Professor 

Ralph S. Baric3 

Associate Professor 

Mark D. Sobsey 1 

Professor 

1Department of Environmental Sciences and Engineering 

3Department of Epidemiology 

School of Public Health 

University of North Carolina at Chapel Hill 

Chapel Hill, NC 27599 

2Department of Pathology 

Tulane Medical Center 

New Orleans, LA 70112 

INTRODUCTION 

The documented evidence of outbreaks of enteric viral 

disease by consumption of contaminated water has increased 

significantly in the past decade (Craun, 1988). Reports of 

enteric virus isolations from drinking water sources and 

from finished water provide evidence for an increased need 

for periodic or perhaps even routine monitoring of these 

waters for viral contamination (Gerba and Rose, 1990). 

Enteric viruses have been isolated on at least 37 occasions 

from treated drinking water in the united States and around 

the world (Bitton et al., 1985; Gerba and Rose, 1990), some 

of which met quality standards for indicator bacteria and 

other parameters (Deetz et al., 1984; Gerba et al., 1984, 

1985 and 1986). Unfortunately, the methods used for human 

enteric virus isolation from water rely on animal cell 

culture and are technically difficult, time consuming and 

relatively expensive to conduct. In addition, current 

indicator bacteria inadequately reflect the presence of 

waterborne enteric viral pathogens. It has been clearly 

established that enteric viruses are more persistent in the 

aquatic environment and more resistant to water and 

wastewater treatment processes, including disinfection, 

than are fecal indicator bacteria (Hoff I 1986; Leong I 

1983). Widespread and routine monitoring of wastewater 

effluents or raw and finished drinking water for human 

enteric viruses will require rapid, sensitive and 

inexpensive methods. New virus detection methods based on 

the use of gene probes (nucleic acid hybridization) and 

polymerase chain reaction (target nucleic acid in-vitro 

amplification) for detection of specific enteric viruses 

or closely related groups of viruses show considerable 

De Leon 833 
promise as inexpensive and rapid methods for virus 

detection in the environment. 

Gene probes and other nucleic acid techniques have recently 

been used for the detection of enteric viruses in 

environmental samples. Gene probe hybridization to genomic 

viral RNA has been reported to detect 2.5 X 105 physical 

particles of rotavirus SA-11 (Dimitrov et al., 1985), 104 

physical particles of hepatitis A virus (Jiang et al., 

1986), and 500-1000 PFU of rotavirus SA-11 and coxsackie 

B3 (De Leon, 1989; Shieh et al., 1990). Margolin et al., 

(1986) have reported the detection of 1-10 PFU of 

pOliovirus using probes with a specific activity of 2 X 109 

CPM/ug of DNA. In general, RNA probes are 5-8 fold more 

sensitive than DNA probes (Jiang et al., 1987; Shieh et 

al., 1990; Dubrou et al., 1990). Although most reports on 

detection sensitivities are 100 fold less than a desired 

goal of 1-10 infectious units, several investigators have 

reported detection of enteric viruses in environmental 

samples. Margolin et al., (1986), using a cDNA probe to 

poliovirus, detected enteroviruses in environmental 

samples, and these samples also were enterovirus positive 

by a modified cell culture assay (De Leon and Gerba, 1985). 

In a later study, Margolin et al., (1989) used pOliovirus 

and hepatitis A gene probes to detect enteroviruses in 

groundwater. Jiang et al., (1986) have used cDNA probes to 

detect seeded hepatitis A in estuarine samples with a sensitivity of 10 particles and in a subsequent study they 

detected naturally occuring hepatitis A viruses in 

estuarine samples using ssRNA probes (Jiang et al., 1987). 

All virus detections in environmental samples by gene 

probes thus far reported have been made with radioactively 

labeled probes. Non-radioactive detection systems are 

approximately ten times less sensitive (Jiang et al., 1987) 

and thus not directly applicable for detecting the low 

number of viruses expected in environmental samples. 

However, the sensitivity of gene probes in environmental 

samples can be increased by the use of other molecular 

techniques in conjunction with gene probe hybridization. 

Recent advances in nucleic acid detection by gene probes 

coupled with target nucleic acid amplification techniques 

such as polymerase chain reaction (PCR) offer exquisite 

sensitivity and detection specificity of enteric viruses 

(Shieh et al., 1990) and other microbial agents in the 

environment. Polymerase chain reaction (PCR) provides a 105

-106 enrichment of a specific nucleic acid sequence and 

is capable of detecting a single copy of DNA (Ou et al., 

1988; Saiki et al., 1988) . Although Thermus aguaticus (Taq) 

polymerase cannot use RNA as a template, the PCR technique 

is adaptable to the amplification of RNA viruses by a 

simple reverse transcriptase reaction prior to polymerase 

chain reaction (RT-PCR). Recently, this approach has been 

used for the amplification of enteroviruses (Hyypia et al. , 

1989; Rotbart, 1990; Chapman et al., 1990; Shieh et al., 

1990), hepatitis A virus (Jansen et al., 1990) and 

rotaviruses (Gouvea and Glass, 1990). 

834 
The choice of primers for PCR amplification of specific 

gene sequences in target viral genomes is crucial for the 

efficiency and the specificity of the method in detecting 

particular viruses and virus groups. Based on genetic and 

antigenic information it should be possible to select three 

sets of primers that will make it possible to detect nearly 

all of the human enteroviruses, hepatitis A virus and the 

group A rotaviruses. Some of the primers used for RT-PCR 

amplification of enteroviruses were selected from the 5' end 

non-coding region of their genomes. These regions contain 

highly conserved sequences that permit the development of 

pan-specific primers and probes capable of detecting a variety of different enteroviruses, including the 

polioviruses, group A and B coxsackieviruses and 

echoviruses (Tracy, 1988). Hepatitis A virus is unrelated 

in nucleotide sequence to other enteroviruses, and separate 

amplification primers were selected from both it's 5' end 

and specific capsid regions which are highly conserved 

among strains isolated from different parts of the world 

(Cohenet al., 1987; Gust and Feinstone, 1988). Because 

sequence information for human rotaviruses is more limited, 

primer selection was based on a commercially available 

primer set. 

The objective of this study was to develop and evaluate the 

basic methodology needed for the detection of 

enteroviruses, hepatitis A virus and rotavirus in 

environmental samples by the combined use of reverse transcriptase-polymerase chain reaction and nonradioactively labeled oligoprobes. 

MATERIALS AND METHODS 

VIRUSES AND CELLS 

Enteroviruses were propagated in A549 (human lung carcinoma 

-derived) or BGMK (Buffalo green monkey kidney) cells and 

assayed for infectivity by plaque assay (Sobsey et aI, 

1978). HAV, strain HM175, (NIH prototype strain) was grown 

in FRhK-4 (fetal rhesus kidney-derived) cells. other 

strains of HAV (CR326 and HAS 15) were propagated in A549 

cells. Viruses were harvested from infected cell lysates 

by freeze-thawing, Freon extraction and polyethylene glycol 

precipitation. HAV was further purified by equilibrium 

centrifugation in CsCl gradients and then rate-zonal 

centrifugation in sucrose gradients. Rotaviruses were 

propagated in MA-I04 (rhesus monkey kidney) cells (Sato et 

al., 1981) and assayed for infectivity by plaque assay (SAIl) or immunofluorescence (Human rotavirus Wa)(Smith and 

Gerba, 1982). 

PRIMERS AND PROBES 

Sequence analysis of several partial or fully sequenced 

enteroviruses indicates several highly conserved regions 

in the 5' end of the enterovirus genome (Iizuka et al., 

1987; Jenkins et al., 1987; Tracy et al., 1985; Hughes et 

al., 1988; Pilipenko et al., 1989; Hughes et al., 1989; 

Toyoda et al., 1984). Some of these regions are also 

conserved in the closely-related rhinoviruses and bovine 

enteroviruses. Primers selected from conserved regions in 

835 
human enteroviruses but not found in the rhinoviruses nor animal enteroviruses would be most appropiate for 

environmental samples. The downstream or antisense primer 

was selected from a region conserved in the human 

enteroviruses but lacking in the rhinoviruses and bovine 

enteroviruses which have been sequenced (Figure 1) (Jenkinsr 

1987; Earle r J.A.P. et al. r 1988). The downstream 

enterovirus primer (Figure 1) (EV-R 5' -ACCGGATGGCCAATCCAA3') r which was synthesized as single stranded DNA using an automated synthesizer is complementary to the sequences 

comprising nucleotides 627-644 in the coxsackie B virus 

genomic sequence (Jenkinsr et al., 1987; Iizuka et al., 

1987). The upstream enterovirus primer (EV-L 5' 

CCTCCGGCCCCTGAATG-3') was synthesized in the (+) sense orientation from the sequence of the coxsackievirus B 

genome and comprises nucleotides 449-465. A strongly 

conserved region in the intervening sequences between the 

primers was chosen as an oligonucleotide probe. The probe 

comprises sequences 545-567 (EV-In 5'-

ACTACTTTGGGTGTCCGTGTTTC- 3') of the coxsackievirus B genome 

and was synthsized in the (+) -sense orientation, thus 

capable of hybridizing only with cDNA or PCR product and 

not with viral genomic RNA. 

Primer selection for hepatitis A virus was based on the 

HM175 strain. Two regions of the genome that exhibit high 

sequence conservation, the 5' non-coding sequences and the 

VP3 capsid protein, were used (Cohen 1987; Gust and 

Feinstone, 1988). The downstream 5' non-coding region 

primer for HAV (Table 1) (HAV-5 'R 5' -CGGCCAGAGCCTAGGGCAAG3') is complementary to the sequence comprising nucleotides 

243-262 of HM175. The upstream primer for the 5' non-coding 

region of HAV (HAV-5'L 5'-CCTCTTGGAAGTCCATGG-3') was synthesized in the (+) -sense orientation and comprises 

sequences 33-50 of the HM175 HAV strain. The internal probe 

for the resulting amplified fragment comprises sequences 

117-134 (HAV 5'-In). The downstream primer in the capsid 

region of HAV (HAV-CR 5'-CTCCAGAATCATCTCC-3') is the 

complementary sequence to nucleotides 2211-2226 of the 

HM175 strain and the upstream primer in the capsid region 

(HAV-CL 5' -ACAGGTATACAAAGTCAG-3') is in the (+) sense 

orientation. The internal probe comprises sequences 2170-

2191 (HAV C-In 5'-TTGCTCCTCTTTATCATGCTAT-3'). 

The rotavirus primers were obtained from a commercial 

supplier (GENEMED, Biotechnologies Inc., San Francisco CA 

94080). The supplier derived the primers from segment 6 of 

the human rotavirus (HRV) Wa strain genomic sequence. The 

downstream primer (HRV-R 5'-ACAGACTTTCATTTGCGTCCGCAA-3') 

is complementary to sequences 1028-1051 and the upstream 

primer (HRV-L 5'-ATAGAAGACAGCGCACCGGATTTG-3' is homologous 

to sequences 451-474. The internal probe, also provided by 

GENEMED, comprises sequences 631-670 of HRV Wa (HRV-In 5'-

CGAACATACAGCAATTTGAACATATCGTCCAGCTTAGGCG-3') (Both et al., 

1984) . 

REVERSE TRANSCRIPTION AND ENZYMATIC AMPLIFICATION CRT-PCR) 

Procedures based on single tube reverse transcription and 

polymerase chain reactions were used preferentially. The 

836 
method of Gouvea and Glass (2990) was tested with the three 

virus groups: enteroviruses, HAV and rotaviruses. This 

method utilizes 2X Tag buffer for both the reverse 

transcription and the enzymatic amplification and includes 

7% DMSO in the reaction. Tag polymerase and Tag buffer from 

Promega ( 2 X Tag buffer= 50 mM KCI, 20 mM Tris-HCI pH 9.0 

at 25°C, 2.5 roM MgCl2 , 0.02% gelatin and 0.2% Triton X-200) 

were used with this method. The RNA-PCR kit from Perkin 

Elmer-Cetus (Norwalk, CT) was tested with polioviruses 

following manufacturer's instructions, with the exception 

that the reaction volume during reverse transcription was 

increased from 20 to 30 ul to accomodate a larger sample 

volume. The downstream primer was used during reverse 

transcription by the Gouvea and Glass method for both HAV 

and other enteroviruses. In the case of rotaviruses, cDNA 

synthesis was initiated with the upstream and downstream 

primer pair because rotaviruses have a dsRNA genome. When 

using the Perkin Elmer-Cetus RNA PCR kit, the random 

primers provided were used for the reverse transcription 

reaction. 

The reaction cocktail was prepared in a single tube, mixed 

and dispensed into individual tubes for all samples within 

a single trial. The reaction cocktail consists of 20 ul of 

20X tag buffer, 20 ul of a 2.5 roM nucleotide mixture, 7 ul 

of DMSO, 60 ul of deionized water, and 2 nanomoles of 

downstream primer. Each tube was overlayed with 200 ul of 

mineral oil (Sigma, st. Louis, MO) and sample added 

underneath the oil in a 20 ul volume. The tubes were heated 

at 99°C for 5 min in the thermal cycler, chilled on ice for 

2 min, centrifuged briefly, and then 5 units of AMV reverse 

transcriptase and 20-20 units of RNasin were added 

underneath the oil. Samples were added to the thermal 

cycler with a preheated block at 42°C and incubated for one hour. After cDNA synthesis, sample tubes were heated to 

99°C for 5 min to completely denature the reverse 

transcriptase. Aft·er brief centrifugation , additional 

primers and 5 units of Tag polymerase were added to each 

tube. Samples were amplified in a Perkin Elmer thermal 

cycler for 40 cycles. The cycle sequence was as follows: 

95°C for 2.5 min, 49 or 55°C for 2.5 min, and 72°C for 2.5 

min. Samples were kept on ice until the block reached 95°C. 

OLIGOPROBES AND PCR PRODUCT HYBRIDIZATION 

Oligoprobes were 3 I labeled with digoxigenin-UTP using 

terminal transferase and purified by ethanol precipitation 

following instructions in the BMB Genius non-radioactive 

end labelling kit (Indianapolis, IN). PCR reaction products 

(2.5-20 % of reaction volume) were mixed with 90 ul of 2 

X SSC, boiled for 5 min, chilled on ice and slot-blotted 

on nylon membranes (DuPont, Boston, MA). The blotted 

samples were bound to the membranes by UV crosslinking 

using shortwave UV light for 3 min at a distance of 25 cm. 

prehybridization and hybridization were performed following 

BMB non-radioactive detection system specifications. 

Membranes were prehybridized for 4 hrs and hybridized 

overnight with 5-20 ng of digoxigenin labeled oligomer per 

mL of solution. The blots were washed twice with 6X SSC at 

837 
room temperature and three times with 6X SSC at Tm-20oC of 

the oligo PCR product hybrid. 

ENVIRONMENTAL SAMPLE PROCESSING FOR PCR 

Eight liters of a raw sewage sample were concentrated ~OOfold by adsorption to an electropositive 30S filter and 

eluted with beef extract following conventional methodology 

(AWWA, ~990). The sewage concentrate was fluorocarbon 

extracted, filter sterilized through a 0.2 um pore size 

filter (pretreated with beef extract) and seeded with an 

equivalent of 500 PFU of poliovirus for each subsequent PCR 

reaction. The virus seeded raw sewage concentrate 

constituted 70% v/v of the final PCR reaction. Sephadex G200 (Pharmacia, Piscataway, NJ) spun columns were prepared 

according to Maniatis (~982) except that silane treated 

glass wool (Supelco, Bellafornte, PA) was used to plug the 

column and the centrifugal force was decreased to 400 x g 

to avoid crushing the column. Column volumes of ~.5 and 2.0 

mL were used to process 0.25 mL of seeded sewage 

concentrate. Spun column excluded volume effluents were 

used directly for RT-PCR. 

RESULTS 

PRIMER SELECTION 

The detection of enteroviruses, hepatitis A virus and 

rotaviruses has been targeted because the human 

enteroviruses represent excellent candidate indicator 

viruses for fecally contaminated water and both HAV and 

rotaviruses are significant pathogens documented to be 

transmitted by drinking water. Sequence comparison of the 

5' non-coding region (740 bases) of poliovirus typees ~, 

2 and 3 (Toyoda et al., ~984), coxsackie viruses A 2~ 

(Hughes et al., ~989), B ~, 3 and 4 (Iizuka et al., ~987; 

Tracy et al., ~985; Jenkins et al., ~987), rhinovirus B~ 

(Hughes et al., ~988) and a bovine enterovirus (Earle et 

al., ~988) indicate strongly conserved regions from which 

amplification primers and specific oligo-probes may be 

selected (Figure ~). Strong sequence conservation at 

nucleotides 627-644 is observed between PV ~, 2 and 3, CB 

~ and 3 and CA2~, whereas the bovine enterovirus exhibits 

strong divergence in this region. Two other sections with 

strong sequence conservation within the picornaviruses can 

be readily identified at nucleotides 449-475 and 536-567 

(Figure ~). The upstream primer has been selected at 

sequences 449-465 and the internal probe at sequences 545-

567. The amplified segment is ~96 base pairs long. 

Previous studies indicate that the 5' non-coding and the 

VP3 capsid protein regions are highly conserved among 

different HAV strains (Cohen et al., ~987 i Ticehurst, 

~988). To detect HAV, a primer pair from each conserved 

region was selected. The ability to amplify HAV from two 

regions could potentially be used as a confirmatory test. 

In the 5' non-coding region the downstream primer comprises 

nucleotides 243-262 and the upstream primer nucleotides 33-

50. In the VP3 capsid protein region nucleotides 2020-2037 

and 22~~-2226 were used as a primer pair. Commercially 

838 
available rotavirus primers derived from segment 6 of the 

Wa strain were used (Table 1). 

RT-PCR AMPLIFICATION OF ENTEROVIRUSES. HAV AND ROTAVIRUSES 

Previous work by our group (Shieh et ala I 1990) has 

demonstrated that the pan-enterovirus primer pair selected 

can amplify 17 of 17 enteroviruses tested (including 

poliovirus 1, coxsackieviruses A 5, 9, 12, and 17, 

coxsackieviruses B 3 and 4, echoviruses 1, 2, 4, 5, 7, and 

12) when the primer annealing temperature is kept at 45°C. 

Hepatitis A virus was not amplified by these primers. The 

pan-enterovirus oligoprobe also detected all 17 

enteroviruses when both hybridization and wash temperatures 

were kept at Tm -20°C. 

The pan-enterovirus primers were used to amplify poliovirus 

1 and coxsackievirus B3 under various conditions. The use 

of random prlmlng during reverse transcription was 

investigated because this could potentially convert all RNA 

viruses in an environmental sample into the more stable 

cDNA in a single reaction. Reverse transcription primed by 

random hexamers followed by PCR amplification resulted in 

some non-specific amplification of CB3 RNA extracted from 

cells if the annealing temperature during PCR was 45°C. 

When poliovirus 1 virions and a pan-entrovirus primer 

annealing temperature above 49°C were used, only the 

expected 196 bp band was observed, even after 40 cycles 

(Figures 2A and 3A). These pan-enterovirus primers worked 

effectively in amplification cocktails containing either 

no DMSO or 7% DMSO. Twelve percent DMSO worked only if the 

tubes were not overlaid with mineral oil during the initial 

heating of the sample. Amplification from CB3 cellularderived RNA was also succesful when all reaction components 

were added before the RT reaction and then proceeded 

through PCR amplification with the omission of a 5 min 

heating at 99°C between RT and PCR amplification (data not 

shown) • 

Detection of HAV by RT-PCR with the 5' end non-coding 

region primer pair was succesfull but more difficult than 

for other enteroviruses. The use of 7% DMSO in the reaction 

cocktail was necesary and only moderate yields of PCR 

product were obtained (Figure 2A). The amplification 

products are not always "clean" and specificity of 

amplification varied among strains. HAV amplification was 

more successful from the VP3 capsid protein. An amplified 

fragment of 207 bp is readily obtained when primers derived 

from nucleotides 2020-2037 and 2211-2226 were used (Figure 

2A and Table 1). Comparisons between the 5' non-coding and 

VP3 region primer sets indicate that the capsid region 

primers may also result in greater sensitivity (Figure 2A) . 

An amplified fragment of about 601 bp in length is readily 

obtained from the Wa strain of rotavirus using the 

commercial primer pair (Figure 2A). Nonetheless, no specific amplification of three group A animal rotaviruses 

(the simian strain SA-11, the bovine strain NCDV-1 and a 

porcine rotavirus natural isolate) and eight group A human 

isolates (2 each from the four known human serotypes) was 

839 
~~<\ 

obtained using the commercial rotavirus type A primer pair 

under the same conditions (Data not shown). RT-PCR of these 

viruses was unsuccessful even with a primer annealing 

temperature of Tm -20°C. When amplifying at the low 

annealing temperature,. a smaller, secondary band 

(approximately 220 bp long) can be observed with Wa 

rotavirus and with some of the human rotavirus isolates. 

The need to select alternate rotavirus primers which can 

amplify human rotaviruses and, hopefully, exclude animal 

rotaviruses is evident. 

SIMULTANEOUS AMPLIFICATION OF POLIOVIRUS r HEPATITIS A VIRUS 

AND ROTAVIRUS 

The amplification of enteroviruses, HAV and rotaviruses in 

a single PCR run is a practical approach to determine the 

presence of viruses in a sample. To test the feasibility 

of this approach, HRV Wa, PV1. and HAV were each amplified 

in a separate tube with their respective primer pair. RTPCR amplification of the three viruses was readily obtained 

in a single PCR run when the lowest annealing temperature 

(49°C) of all the primer sets was used (Figure 2A, Table 

1.). The expected amplified fragments were obtained for HRV 

Wa (601. bp), PV1. (1.96 bp), HAV in the capsid region (207 

bp) and HAV in the 5' non-coding region (230 bp). Although 

each virus was amplified in a separate tube, it is evident 

from these results that multiple viruses can be detected 

simultaneously. The amplification of multiple virus groups 

in a single tube reaction may also be feasible, but its 

effectiveness will have to be determined empirically. When 

an annealing temperature of 49°C is used to amplify HRV Wa, 

a smaller secondary band is frequently obtained which is 

not observed when annealing temperatures are above 55°C. 

Single and distinct bands were obtained for PV1. and for 

HAV. Amplification from the 5' end of HAV was less 

efficient than in the capsid region. The selection of 

multiple primer sets with similar Tm and non-complementary 

sequences and the optimization of RT-PCR conditions may 

lead to multiple virus group amplification in a single tube 

reaction. 

The PCR products obtained from the simultaneous 

amplification of HRV, PV1., and HAV were direct blotted 

(1./40th of the reaction products) in four replicate 

membranes. In addition to the amplified fragments, lambda 

DNA (250 ng per slot) as a non-specific probe binding 

control and 1.00ul of the diluent as a negative control were 

blotted. The negative control for the HRV amplification was 

blotted on the membrane to be probed by the respective HRV 

oligoprobe and so forth for the other 3 membranes. Also SA1.1. rotavirus segment 6 cDNA was blotted in the membrane to 

be probed by the HRV oligoprobe, coxsackievirus B5 5' end 

cDNA (nucleotides #1.-747) (Shieh et al., 1.990) as control 

for the pan-enterovirus probe, and HAV 5' cDNA (nucleotides 

#1.62-848) (Shieh et al., 1990) as control for the 5' end 

HAV probed membrane. Each membrane was hybridized with a 

non-radioactively labeled HRV Wa, pan-enterovirus, HAV 

capsid, or HAV 5' internal oligomer at Tm -20°C (Figure 

2B). The results of this experiment clearly indicate that 

each oligomer probe was highly specific and hybridized only 

840 
to cDNA or to the amplified fragment having complementary 

sequences, but not to the negative controls. The only cDNA 

control which could be detected by any of the four oligomer 

probes was the cDNA from the coxsackievirus B 5' end 

sequences #1-747. The Wa rotavirus probe has 12 base 

mismatches with the equivalent region in SA-11. The 5' HAV 

oligoprobe sequences (117-134) are not included in the HAV 

cDNA blotted (162-848). These data clearly indicate that 

simultaneous detection of several virus groups is possible. 

SENSITIVITY OF RT-PCR AND NON-RADIOACTIVE OLIGOPROBES 

The sensitivity of two RT-PCR procedures was determined 

with PV1. The major diff·erence between the two procedures 

tested is the reverse transcriptase reaction conditions. 

One procedure utilizes 1 X Tag buffer (low magnesium 

concentration), the downstream (3') primer, and 7% DMSO in a 100 ul reaction volume. This procedure resulted in 

detection of 0.5 PFU of poliovirus as determined by uv 

visualization of ethidium bromide-stained gels (Figure 3A) • 

A second RT-PCR procedure, with a comercial kit (Perkin 

Elmer-Cetus), has a high magnesium concentration buffer, 

random primers and an adjusted 30 ul reaction volume for 

the reverse transcription reaction. The sensitivity of the 

RT-PCR commercial kit is 10 fold greater than the previous 

method (0.05 PFU of poliovirus) (Figure 3A). 

Direct slot-blotting of 10 ul of RT-PCR reaction products 

from the poliovirus sensitivity determinations followed by 

non-radioactive, pan-entero, oligoprobe hybridization 

clearly demonstrates specific hybridization to probe at the 

same level of sensitivity as obtained by UV visualization 

of the ethidium bromide stained gel (also with 10 ul of 

reaction) (Figures 3A and B). Non-specific reactions were 

not observed between the pan-entero probe and PCR amplified 

fragments of HRV or HAV. In addition, non-specific 

hybridization signals were not observed with heat denatured 

viral particles (10 fold dilutions 500-.005 PFU) spoted on 

the same membrane. The pan-enterovirus oligoprobe sequence 

is homologous to viral genomic RNA, thus it only hybridizes 

with the complementary DNA sequences obtained in RT-PCR. 

The intensity of the signal observed in figure 3B was 

obtained in less than 2 hrs of colorimetric reaction. RTPCR amplification of target viral RNA followed by nonradioactive oligoprobe detection produces sharp end-point 

titrations conducive to most probable number (MPN) 

estimates of virus numbers in samples. 

ENVIRONMENTAL SAMPLE CLEAN UP FOR RT-PCR 

Simple and direct virus purification methods compatible 

with RT-PCR detection are needed for processing of 

environmental samples. Preliminary experiments in sample 

clean-up using Sephadex G-200 spun colUmns and divalent 

cation chelation by EGTA on a 100 fold raw sewage 

concentrate seeded with 500 PFU of poliovirus indicate that 

this approach is very promising (Figure 4). No RT-PCR 

amplification was observed in the PV seeded concentrate 

which received no treatment (lane A) or RT-PCR in 2 mM EGTA 

(lane B). Passage of sample through a 1.5 mL G-200 Sephadex 

column (6:1 column to sample volume) and RT-PCR in 2 mM 

841 
EGTA results in a PCR amplified fragment of the appropiate 

size (lane C). Passage of sample through a 2.0 mL Sephadex 

G-200 column (8:1 column to sample volume) without EGTA 

results in a large amount of non-specific amplification 

(lane D). The combined use of a 2.0 mL column and RT-PCR 

in 2 roM EGTA gave the desired PCR product as a single band 

(lane E). An overall 85% recovery of poliovirus, hepatitis 

A virus and rotavirus was obtained in the excluded volume 

from the Sephadex G-200 spun columns (data not shown). 

DISCUSSION 

RT-PCR linked to non-radioactive oligoprobes is a very 

promising method for monitoring of drinking water for 

important human enteric viruses, including enteroviruses, 

HAV and human rotaviruses, because it offers exquisite 

sensitivity as well as specificity of virus detection. In 

order to be useful, the primers and the reaction conditions 

selected for RT-PCR of specific groups of enteric viruses 

should: (a) give specific amplification of the target 

viruses, (b) detect these viruses at the level of 

sensitivity required, (c) and provide reproducible results. 

The selection of pan-enterovirus primers which amplify 

human enteroviruses and exclude hepatitis A viruses, animal 

enteroviruses, rhinoviruses and other members of the 

Picornaviridae family would be most useful for monitoring 

water for human fecal contamination. The pan-enterovirus 

primer set and perhaps the HAV capsid primers best fulfill 

these criteria. The results obtained in this study suggest 

that the pan-enterovirus primers used are good candidates 

for detection of human enteroviruses in environmental 

samples. However, extensive sequence analysis, testing of 

a larger number of enteroviruses, more widespread 

crossreactivity studies with other enteric viruses and 

other microorganisms encountered in water are needed to 

fully assess the adequacy of these pan-enterovirus primers 

for use with environmental samples. Other investigators 

have also selected primers in the 5' non-coding region of 

the picornavirus genome for the amplification of 

picornaviruses (Hyypia et al., , 1989) enteroviruses and 

rhinoviruses (Rotbart, 1990; Gama et al., 1989) and 

enteroviruses (Chapman et al. I 1990). The majority of these 

investigators have selected their downstream primers closer 

to the 5' end of the genome in regions of greater sequence 

conservation with rhinoviruses and bovine enteroviruses 

(approximate map positions #443-468 [Hyypia), 547-562 

[Gama], 584-603 [Rotbart]) than our downstream primer. 

Rhinoviruses can be excluded from amplification by 

selecting the downstream primer (used for the initial reverse transcription reaction) within the last 100-120 

nucleotides of the 5' non-coding region of the 

enteroviruses, because these sequences are not found in the 

rhinoviruses (Tracy, 1988). The primers chosen by Chapman 

et al. (1990), which are within a few nucleotides from the 

primers we have selected, successfully amplified 40 of 41 

enteroviruses and the amplified products were detected by an internal oligomer similar to ours. Echovirus 22 was the 

only member not amplified by Chapman et al., which provides 

further evidence to a previous suggestion that it may not 

842 
belong to the enterovirus group (Rotbart et al., 1988). A 

compilation of the 41 enteroviruses tested by Chapman et 

al. and the 17 previously tested by our group results in 

47 distinct members (66% of all enteroviruses) successfully 

tested in RT-PCR using similar primers. 

Sequence data for HAV shows that the 5' non-coding region 

and the VP3 capsid region are highly conserved among 

diverse HAV strains (Ticehurst, 1989 i Cohen,1987). From our initial evaluation, a primer set in the 5' non-coding 

region of the HAV genome (nucleotides 33-50 and 243-262) 

did not work very effectively with all strains of HAV. Most 

likely this is due to the predicted RNA secondary structure 

between nucleotides 1-41 of the HAV genome and not to 

sequence heterogeneity (Cohen et al., 1987). Selection of 

primers downstream of nucleotide 41 may lead to better 

amplification, but overall RT-PCR of HAV appears to be 

difficult in the 5' non-coding region {Jansen, 1990}. 

Amplification of HAV from the VP3 capsid region using 

primers for nucleotides 2020 to 2037 and 2211 to 2226 

worked consistently. Thus, this region may be a better 

candidate for development of RT-PCR methodology for use by 

water utilities. 

The amplification of rotaviruses with the commercial 

primers derived from segment 6 was successful, in our hands, only with HRV Wa strain and failed to amplify eight 

other human rotaviruses (2 of each serotype). Sequence 

information for rotavirus is still limited but segment 4 

may be a better genome location for detection of human 

rotaviruses (Flores, 1989). A cDNA probe of segment four 

of rotavirus Wa has been used to detect rotaviruses in 

environmental samples, and it did not exhibit 

crossreactivity to SA-11, NCDV-1 and a porcine rotavirus 

isolate (De Leon, 1989). Kantharidis et al., (1987) found 

significant sequence variation in segments 4 of human 

rotavirus strain RV-5 and simian rotavirus SA-11. In 

addi tion gene 4 may be used to distinguish between more 

virulent strains of human rotavirus from those which may 

produce asymptomatic infections (Taniguchi et al., 1989). 

Single tube RT-PCR methods differ in buffer composition, 

type of reverse transcriptase, primer used for first strand 

cDNA synthesis, and volume of the RT reaction. Of the two 

methods we tested, the commercial RT-PCR kit resulted in 

a ten-fold greater sensitivity in detection than a generic 

cocktail, with a positive result for as little as 0.05 PFU 

of pOliovirus (approximately 5-50 molecules of RNA). The 

kit has been optimized for the reverse transcription 

reaction by use of higher magnesium concentration, random 

primers and smaller reaction volume (20 ul vs 100ul). 

However, for testing of environmental samples the volume 

of the RT reaction may need to be increased and the 

potential impact of this increased volume on the overall 

sensitivity of the assay must be investigated in future 

studies. 

The non-radioactive oligoprobe labeling and detection 

system used in this study provides the needed sensitivity 

843 
for detecting PCR products. strong colorimetric reactions were observed within one hour after the probe was 

hybridized to the target sequences and the digoxigenin 

specific antibody was bound to the probe. At the limit of 

sensitivity of virus detection by the RT-PCR methods used, 

the non-radioactive probe provided detection within one 

hour at the titration end point detected by gel 

electrophoresis of PCR products. 

Our preliminary investigations of environmental sample 

clean-up methods indicate that gel chromatography can be 

adapted to the removal of RT-PCR-interfering factors. So 

far, only one type and grade of gel filtration material 

(Sephadex G-200) has been tested. clearly, a more thorough 

and systematic evaluation of other gel chromatography 

materials and grades, their combinations, optimization of 

gel bed volume to sample ratios, equilibration and running 

buffers, and potential divalent ion chelators is necesary 

in order to obtain an optimum clean-up method for routine use on environmental samples. Such investigations are 

proceding in this laboratory. The RT-PCR-oligoprobe method 

is a very promising non-conventional approach to the 

detection of enteric viruses in the envinronment. 

REFERENCES 

Bitton, G., Farrah, S.R., and Montague, C., Binford, M.W., 

Scheverman, P.R., and Watson, A. Survey of Virus Isolation 

Data from Environmental Samples, EPA Contract No 68-03-

3196, 1985, Cincinnati, OH. 

Chapman, N.M., Tracy, S., Gauntt, C.J., and Fortmueller, 

U. 1990. Molecular detection and identification of 

enteroviruses using enzymatic amplification and nucleic 

acid hybridization. J. Clin. Microbiol. 28:843-850. 

Cohen, J.I., Ticehurst, J.R., Purcell, R.H., Buckler-White, 

A., and Baroudy, B.M. 1987. Complete nucleotide sequence 

of wild-type hepatitis A virus: Comparison of different 

strains of hepatitis A virus and other picornaviruses. ~ 

Virol 61:50-59. 

Craun, G.F. Surface Water Supplies and Health, Journal of 

American Water Works Association, 1988, 80: 40-52. 

Deetz, T.R., Smith, E.M., Goyal, S.M., Gerba, C.P., Vollet, 

JJ., Tsai, L., DuPont, H.L., and Keswick, B.H. 1984. 

Occurrence of Rota- and Enterovirus in drinking water and 

environmental water in a developing nation. Wat. Res. 

18:567-571. 

De Leon, R. and Gerba, C.P. Enhancement of the Sensitivity 

of the BGM Cell Line to Enteroviruses, ASM Meeting, 1985, 

Washington, DC. 

De Leon, R., Use of Gene Probes and an Amplification Method 

for the Detection of Rotaviruses in Water, PH. D. 

Dissertation, University of Arizona, 1989, Tucson, AZ. 

844 

I 
Dimitrov, D. J., Graham, D.Y., and Estes, M.K., Detection 

of Rotavirus by Nucleic Acid Hybridization with Cloned DNA 

of Simian Rotavirus SA-II Genes, Journal of Infectious 

Diseases, 1985, 152:293-300. 

Dubrou, S., Kopecka, H., Lopez-pila, J.M., Marechal, J., 

and Prevot, J., Detection of Hepatitis A Virus and other 

Enteroviruses in wastewater Samples by Gene Probe Assays, 

International svmposhim on Heal th-Relat.ed water 

Microbiology, 1990, Tubingen, Germany. 

Gama, R.E., Horsnell, P.R., Hughes, P.J., North, C., Bruce, 

C.B., Al-Nakib, W., and Stanway, G. 1989. Amplification of 

rhinovirus specific nucleic acid from clinical samples 

using the polymerase chain reaction. J. Med. Virol. 28:73-

77. 

Gerba, C.P., Keswick, B.H., DuPont, H.L. and Fields, H. 

A. 1984. Isolation of rotaviruses and hepatitis A virus 

from drinking water. Monogr. Virol. 15:119-125. 

Gerba, C.P., Rose, J. R. and singh, S.N. 1985. Waterborne 

gastroenteritis and viral hepatitis. CRC. crit. Rev. 

Environ. Control 15:213-236. 

Gerba, C.P., Rose, J .B., De Leon, R., Toranzos, G.A., 

Singh, S.N. and Keswick, B.H. 1986. Isolation of rota- and 

entero- viruses from three different drinking water 

supplies. Proceedings Water Quality Technology Conference. 

Houston. TX. AWWA. pp. 451-459. 

Gerba, C.P. and Rose, J.B. Viruses in Source and Drinking 

Water, in Drinking water Microbiology, Springer-Verlag, 

New York, 1990, pp. 380-395. 

Gouvea, V., Glass, R.I., Woods, P., Taniguchi, K., Clark, 

H.F., Forrester, B., and Fang, Z. Y., 1990. Polymerase Chain 

Reaction Amplification and Typing of Rotavirus Nucleic Acid 

from Stool Specimens, Journal of Clinical Microbiology, 

28:276-282. 

Gust, I.D. and Feinstone, S.M. 1988. Hepatitis A. CRC 

Press, Boca Raton, FL. 

Hoff, J .C. 1986. Inactivation of Microbial 

Chemical Desinfectants. EPA/600/2-86/067, 

cincinnati, OH. 

Agents bv 

U.S.EPA, 

Hyypia, T., Auvinen, P., and Maaronen, M. 1989. Polymerase 

chain reaction of human picornaviruses. J. Gen. Virol. 

70:3261-3268. 

Jansen, R. 1990. Personal communication. 

J lang, X., Estes, M. K., Metcal f, T. G., and Melnick, J. L. , 

1986. Detection of Hepatitis A Virus in Seeded Estuarine 

Samples by Hybridization with cDNA Probes, Appl. Environ. 

Microbiol., 52:711-717. 

845 
Jiang, X., Estes, M.K., and Metcalf, T.G., 1987. Detection 

of Hepatitis A Virus by Hybridization with Single-Stranded 

RNA Probes, Appl. Environm. Microbiol., 53:2487-2495. 

Leong, L.Y.C. 1983. Review: removal and inactivation of 

viruses by treatment processes for potable water and 

wastewater. wat. Sci. Technol. 15:91-114. 

Maniatis, T., Fritsch, E.F., and Sambrook, J. 1982. 

Molecular Cloning. A Laboratory Manual, Cold Spring Harbor 

Laboratory. 

Margolin, A.B., Hewlett, M.J., and Gerba, C.P. 1986. Use 

of a cDNA Dot-Blot Hybridization Technique for Detection 

of Enteroviruses in Water, Advances in Water Analysis and 

Treatment. AWWA Proceedings, Houston, TX. 

Margolin, A.B., Richardson K.J., De Leon, R., and Gerba, 

C.P. 1989. Application of Gene Probes to the Detection of 

Enteroviruses in water, in Biohazards of Drinking Water 

Treatment, Ed., R.A. Larson, Lewis Publishers, Chelsea, MI. 

Ou, C.Y. et al., science, 1988, 239:295-297. 

Rotbart, H.A. 1990. Enzymatic amplification of the 

enteroviruses. J. Clin. Microbiol. 28:438-442. 

Saiki, R.K., et al., science, 1988, 239:487-491. 

sato, K., Inaba, Y., Shinozaki, R., Fujii, R., and 

Matamuto, M. 1981. Isolation of human rotaviruses in cell 

cultures. Brief report. Arch. Virol. 69:155-160. 

Shieh, Y.S.C., Baric, R.S., Sobsey, M.D., Ticehurst, J., 

De Leon, R., and Walter, R., 1990. Detection of Hepatitis 

A Virus and other Enteroviruses in Water by ssRNA Probes, 

J. Virol. Methods, accepted for publication. 

Smith, E. M. and Gerba, C. P. 1982. Development of a 

method for detection of human rotavirus in water and 

sewage. Appl. Environ. Microbiol. 43:1440-1450. 

Sobsey, M.D., Shields, P.A., Hauchman, F.S., Davis, A.L., 

Rullman, V.A., and Bosch, A. 1988. Survival and persistence 

of hepatitis A virus in environmental samples. In Viral 

Hepatitis and Liver Disease, A.J. Zuckerman (ed.). pp. 121-

124. Alan R. Liss. Inc., New York. 

Ticehurst, J., Cohen, J.I. and Purcell, R.H. 1988. Analysis 

of molecular sequences demonstrate that hepatitis A virus 

(HAV) is a unique picornavirus. In: Viral Hepatitis and 

Liver Disease, A. Zuckerman (ed.). Pp 33-35. Alan R. Liss, 

New York. 

Tracy, S. 1988. The genome of the group B coxsackieviruses. 

In: Coxsackieviruses. A general Update, Eds. M. Bendinelli 

and H. Friedman. Plenum Press, New York. 

AWWA. 1990. Detection of Enteric Viruses, in Standard 

846 
Methods for the Examination of Water and Wastewater, 17th 

Edition Supplement, 1990. 

TABLE 1. PRIMERS FOR ENTERIC VIRUS AMPLIFICATION BY PCR 

Amplified 

Primer Se@ence TmoC* Fragment 

Rotavirus Wa 

Segment 6 

451-474 ATAGAAGACAGCGCACCGGATTTG 72 

601bp 

1028-1051 ACAGACTTTCATTTGCGTCCGCAA 70 

Pan-enterovirus 

5 I Non-coding Region 

449-465 CCTCCGGCCCCTGAATG 58 

196bp 

627-644 ACCGGATGGCCAATCCAA 56 

HeQatitis A virus HM175 

CaQsid Region 

2020-2037 ACAGGTATACAAAGTCAG 50 

207bp 

2211-2226 CTCCAGAATCATCTCC 48 

5 I Non-coding Region 

33-50 CCTCTTGGAAGTCCATGG 56 

230bp 

243-262 CGGCCAGAGCCTAGGGCAAG 64 

* Tm calculated by adding 4°C for every G or C and 2°C for 

every A or T. The Tm of the rotavirus primers was provided 

by the supplier. 

847 
449 46~) 

5 • - CGTCCGGCCCCTGAATG - 3' 

445 502 

AAGAATCG1'ccqGCCCCTGAA.l'GCGGC1'AATCCCI\ACCTCG(;GGC.I\GG,[,(;(;TCi\CAI\ACC 

----G----- ------------------ --------T- -- - f\---- AA- -- - -C --- -G --(;-- ---

-- ---GI: ---------------------------T- T - - - -AT -.- .1\.-- --·-CA-·CTG- - ·-CGG1'-- - - - -- - - - -. - - - - - - - .. -.- - - - - - - - .- - -- .- T - -- -- Tl~ - --A- - - CA -Ace TGGT-G------- -- - --- -- - -------- ------- ----- -T ---- TG- ---- ----CACACCC1'- - - G--

----G----------------------------T----A---A---ATC-C----G-C--

TT--G-------------------------C-TT--.I\-CT-.I\-C--T-GCTCAT~----­

GG1'TT--------AG--,,------1'---------------C-A--G1'·- --CG----- T--

505 (j62 

AG1'GAT1'GGCCTGTCG1'AACGCGGAAG1'CCGTGGGGGAACCGAc;.:rAt..!'(l"['t;(:Gn;TCCGl' 

- ------C--- .--1'- --- ---------------T------.. --------- - --- - ---.- --.- -- .------. ---

--CAGCCA-------------------------------------------------

--G-GGCA-1'G----------G----C--1'-CA------------------ ------- -

--A-GGCA-1'G------ -----G---- -C- --T -(;.1\------- ----- - ---.--- -- -- - --- -- ---- - -. 

-----GGT1'1: I: * -------T---1'------T-------- --------- -----. - --.--- -. -- .. 

-A1'--GCTTATG----- --T-A---- T-G- -G-AT---G------- --- .-... --.- - -- - --.- - - - ---

----1'-GCTACG1'I:--- -----------TG--A---------A --- --- - ---C----.I\"'----

565 b~~ 

G1'T_TGCT1'T1'A1'1'1'1'&A1'1'GTGGCTGCT1'ATGG1'GACAA1'CACAG.I\TTGTTAT\"~I\TAi\AG 

-------G-------T--CA----------------------G-------------"---

-------- - ------C-TGA 11: A--------------------- --- T-- -- .----.- -. -- --- -- -. - .-

-----1\--- -----CC1'-- AG-- - ---.--- - - -- -- -------.-- TGACAGG - - - -. -·C -1'--

-----A--------cc* --AC----- -- --- ------ --------- TGAGI\ F_ - ---.- c- - - - T-

------C-----I\--C--I\C--------------------1'1'I\CGI\I\" -----c -1'-. 

-----AC---T-CC1'T1'A--G'" AAT1'-- ----- - .----- -- -- ·-ATATACI\TI\l;.I\TATATATT -

--------A-1'I\--1'1' --ACI\ACI\A "'T - -. ---- --- - -- --T -G-- T ---AG-AT -G -GTTC . 

625 659 

CGAA1'1'GGA1'1'GGCCA1'CCGGTGI\AI\G1'GI\"GATTCA 3' ['VJ 

------------------ --------G1'- --TGT- F -CAG 3' PV2 

---G----------------A----TG-I\-C-- - ---A- 3' PV:5 

&1'1'------------- --·-----·-CTAACI\" ---G(;I\- 3' CEIl 

-T1I:~--------------------CCAAT---GCT-- 3' C83 

- 1'& --------------C--I\-- - --CTGTGCAAT- --ATT 3' CA~~1 

GC--TCI\1'-GGGT-·- --CAGGTA1'C1' -G-CI\-AATG -1'G 3' R 18 

-CC"--- a ----AAT--TGCC1'--GT-T-ACCTTI\-I\G 3' SF-V 

627 644 

3'-AACGl'AACCGG1'AGGCCA-S' 

FIGURE 1_ Sequence CompRrisons among Enteroviruser-;_ 

Sequences were aligned to the Sabin poliovirus type 

genomic sequence (Toyota, et aJ, J 984) . Hegj OIlS of homol O[~y 

are designated by dashed lines while astericks represent 

either nucleotide insertjons or delet.ions_ PVI-poti.ovirusJ, 

PV2-poliovirus 2, PV3-poliovirus 3, C81-coKsacki0 virus 

Bl,CB3-coxsackie virus [U, CA?l·-coxsackie 1\ 21, RBIrhinovirus 1, BEV-bovine enterovirus_ 

848 
Figure 2A 

Multiple virus RT-PCR. Each virus group has been amplified 

with its respective primer pair in a separate reaction. 

Reverse transcription took place in 1 X Tag buffer from 

Promega, 7% DMSO and the 3' primer (see text). M= markers 

corresponding to 587,540, 504, 458, 434, 267, 234, 213, 

192, 184, 124, 123 (BMB DNA marker V); HRV Wa= human 

rotavirus Wa strain; (-)= complete reaction cocktail 

without virus; PVl= pOliovirus type Ii HAVcap.= hepatitis 

A virus amplified in the capsid regioni HAV5'= hepatitis 

A virus amplified in the 5' non-coding region. The 

amplified fragment sizes are: HRV Wa, 601 bPi PVl, 196 bPI 

HAVcap, 207 bp and HAV5', 230 bp long. 

849 
oligo 

probe 

HRVwa 

Pan-En 

HAVe 

HAVS' 

RT- peR 

> 5t; cL > <: < .- a... I""'" I 

,I 

Figure 2B 

« 

zz 

00"": 

-< V -0 

Direct blotting of PCR products and non-radioactive 

detection by oligoprobe hybridization. The four membranes 

were blotted with 1/40th of the PCR products per slot 

obtained from the multiple virus PCR reaction and with 

various controls. Each membrane was hybridized at Tm -200 

C with either a rotavirus, pan-enterovirus, hepatitis A 

capsid region or hepatitis A 5'non-coding region internal 

digoxigenin labeled oligoprobe. HRV= human rotavirus Wa RTPCR amplified product; PV= poliovirus amplified product; 

HAVc= hepatitis A capsid region amplified product; HAV5'= 

hepatitis A 5' non-coding region amplified product; (-)= 

10 ul of negative control; X DNA= 250 ng of lambda DNA; 

cDNA= for the HRV Wa oligoprobed membrane, 100 ng of SA-II 

segment 6; for the pan-enterovirus probed membrane, 100 ng 

of 5' end cDNA (bp 1-740), for HAVc, no cDNA spotted; for 

HAV5', 100 ng of HAV5' cDNA (bp #162-848). 

850 

j 
Figure 3A 

Sensitivity of RT-PCR for Poliovirus 1. M= markersi PFU 

PVl= 500-0.005 plaque forming units of poliovirus Ii (-)= 

complete reaction cocktail without virus. The virus 

dilutions in the left side of the figure were reversed 

transcribed in 1 X Promega Taq buffer and the downstream 

primer (3'Pr). The virus dilutions in the right side of the 

figure were reversed transcribed with the Perkin-Elmer (PE) 

RT-PCR kit and random primers (Rp). 

851 
RT-PCR Q.. 

Q.. MO 

c.t: COVl 

PFU > w.J CJ~ 

PV1 0- 0- ~O 

500 - ........ SO 

5 - - - - .5 ...... .05 - .005 - (- ) 

I , 

HRV HAV 

PCR product 

Figure 3B. 

sensitivity of RT-PCR and non-radioactive oligoprobe 

hybridization. Poliovirus RT-PCR-derived products were direct blotted (10ul from each dilution) and hybridized 

with digoxigenin-Iabeled pan-entero oligoprobe at Tm-20 C. 

PVl= pOliovirus 1 RNA from 500-0.005 PFU; PE Rp= amplified 

products from the amplification of pOliovirus with the 

Perkin Elmer RNA PCR kit using random primers; Tag B 3'Pr 

DMSO= amplified products from the amplification of 

poliovirus with 1 X Promega Tag buffer, 7% DMSO and the 

downstream primer. HRV HAV PCR products= 10 ul of PCR 

amplified fragments of human rotavirus Wa and hepatitis A 

VP3 capsid region, respectively. 

852 
Figure 4. 

Detection of seeded poliovirus 1 in a raw sewage 

concentrate by RT-PCR. Each PCR reaction contained 500 PFU 

of poliovirus. The sewage concentrate comprised 70% vjv of 

the RT-PCR reaction volume. Lane A= no sample treatment; 

lane B= RT-PCR reaction cocktail with 2 mM EGTA; lane C= 

sample run through a 1.5 mL G-200 Sephadex spun column (6:1 

column to sample volume) and RT-PCR reaction with 2 roM 

EGTA; lane D= sample run through a 2.0 mL G-200 Sephadex 

column (8:1 column to sample volume) before RT-PCR; lane 

E= sample run through a 2.0 mL G-200 Sephadex spun column 

and the RT-PCR reaction cocktail contained 2 mM EGTA. 

853