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Appl Environ Microbiol, March 1998, p. 858-863, Vol. 64, No. 3
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
A Nested Reverse Transcriptase PCR Assay for
Detection of Small Round-Structured Viruses in Environmentally
Contaminated Molluscan Shellfish
J.
Green,1,*
K.
Henshilwood,2
C. I.
Gallimore,1
D. W. G.
Brown,1 and
D. N.
Lees2
Enteric and Respiratory Virus Laboratory,
Virus Reference Division, Central Public Health Laboratory,
Colindale, London NW9 5HT,1 and
Centre for Environment, Fisheries and Aquaculture
Science, Weymouth, Dorset DT4 8UB,2 United
Kingdom
Received 17 July 1997/Accepted 2 January 1998
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ABSTRACT |
We describe the evaluation of a nested reverse transcriptase PCR
(RT-PCR) procedure for the detection of small round-structured viruses
(SRSVs) in molluscan shellfish and the application of this assay for
the detection of SRSVs in commercially produced shellfish and in
shellfish implicated in outbreaks of gastroenteritis. The range of
virus strains detected and the sensitivity of detection were evaluated
by using a representative panel of 21 well-characterized SRSV strains.
The nested RT-PCR detected 15 of 21 SRSVs, demonstrating that the assay
detects a broad range of SRSVs including strains from both genogroup I
and genogroup II. Seeding experiments showed the nested RT-PCR assay to
be 10 to 1,000 times more sensitive than the single-round RT-PCR assay
for the detection of SRSV in shellfish. SRSV-contaminated samples were
identified by nested RT-PCR for shellfish grown in polluted harvesting
areas and for shellfish associated with outbreaks of gastroenteritis
which were negative by a previously described single-round RT-PCR. The
assay was shown to be effective for investigation of virus elimination during commercial shellfish processing procedures such as depuration and relaying and has potential applications for monitoring at-risk shellfish harvesting areas, for investigation of SRSV contamination in
shellfish from producers linked to gastroenteritis outbreaks, and for
the direct detection of virus in shellfish implicated in outbreaks.
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INTRODUCTION |
The small round-structured viruses
(SRSVs) are important human pathogens frequently associated with
gastroenteritis following consumption of sewage-contaminated molluscan
shellfish. Public health controls are hampered by the absence of
methods for the detection of these viruses in shellfish, as they cannot
be grown in tissue culture. Recently, genomic RNA sequences of Norwalk virus (9) and other SRSVs (4, 5, 7, 8, 10, 11, 15-18,
21) have become available and have led to the classification of
SRSVs within the virus family Caliciviridae (3).
The genomic characterization of a number of SRSVs has facilitated the
development of highly sensitive reverse transcriptase PCR (RT-PCR)
assays for the diagnosis of SRSV infection (6, 19). Several
studies have demonstrated the high level of sequence diversity among
SRSVs (1, 20, 23), and this has proved to be the major
obstacle for the development of a diagnostic RT-PCR. Consensus primers which detect all SRSVs have not as yet been identified, but a broadly reactive primer pair which detects approximately 90% of SRSVs
circulating in the United Kingdom (UK) has been described (6). The development of RT-PCR for the direct detection of SRSVs in shellfish has been further hampered by low levels of virus
present in shellfish meat which may also contain potent Taq
inhibitors. We (14) and others (2) have
previously described the development of RT-PCR-based assays for the
detection of SRSVs in molluscan shellfish. Our techniques utilize a
sample extraction procedure optimized for removal of RT-PCR
amplification inhibitors which largely addresses these problems
(12, 14). We have described the application of these
techniques to the detection of SRSVs in shellfish associated with
outbreaks of human disease and in random testing of shellfish sold for
consumption. By a single-round RT-PCR with broadly reactive primers
followed by Southern blot hybridization with a pool of four
digoxigenin-labelled SRSV-specific oligonucleotide probes, SRSVs could
be detected in virtually all oyster samples associated with human
disease and in a small percentage of randomly tested samples. However,
positive results were frequently detectable only through the added
sensitivity of Southern blot hybridization, which indicated that the
RT-PCR was operating at the limits of sensitivity. This hindered
attempts to confirm positives by sequencing of the amplicon and to
genotype the SRSVs detected. This study describes the development and
evaluation of a nested RT-PCR for SRSV which overcomes these
sensitivity limitations and thus facilitates sequencing and other
approaches to RT-PCR amplicon characterization. We describe the
application of this method for the investigation and control of public
health problems arising from consumption of molluscan shellfish.
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MATERIALS AND METHODS |
Viruses.
Evaluation of the range of SRSV strains amplified
by the nested RT-PCR was performed with a panel of 21 fecal samples
which had been shown to contain SRSV by electron microscopy and had been selected to represent the broad range of genomic diversity of
SRSVs. The SRSVs had previously been characterized by sequencing of
small regions of the RNA polymerase following amplification with the
NI-E3 primer pair (6) and/or the SM51-31 and 52-32 primer
pairs (20). The panel comprised 8 genogroup I strains and 13 genogroup II strains. Phylogenetic analysis of polymerase gene
sequences from these strains and published sequences is shown in Fig.
1. Five fecal samples from the panel, two containing genogroup I SRSVs
(panel strains 2 and 5) and three containing genogroup II SRSVs (panel
strains 10, 15, and 18), were used in the comparison of detection
sensitivities of the single and nested RT-PCR assays.
For shellfish seeding experiments, a well-characterized stool sample
shown to contain SRSVs by electron microscopy and by RT-PCR was used
(panel strain 18). Sequencing of a 113-bp region of the RNA polymerase
gene showed it to be a genogroup II strain most closely related to
Mexico virus (10) (Fig. 1). This strain was selected because
Mexico virus-like strains have been only infrequently detected in the
UK in the past 3 years and thus this strain allows the exclusion of
false positives in subsequent investigations of field samples derived
from this positive control sample.
The fecal samples used in these experiments were prepared by making a
1:10 (wt/vol) dilution of stool in phosphate-buffered saline (PBSa;
Dulbecco's formula) followed by thorough mixing and centrifugation at
3,000 × g for 5 min. Supernatants were stored at 4°C
until use. For the comparison of detection sensitivities, further
dilutions from 10
1 to 107 were made with
PBSa. For the seeding experiments, further dilutions of 1:100, 1:500,
1:1,000, 1:5,000, 1:10,000, and 1:50,000 were made with PBSa.
Shellfish.
Commercially purified oysters (Crassostrea
gigas) were used in the seeding experiments. Shellfish were also
obtained from commercial harvesting areas in England and Ireland
subject to varying levels of pollution. Shellfish associated with a
clinical outbreak in Suffolk, England, were obtained from both the
implicated restaurant and the holding tank used for oyster storage
prior to serving. Environmental samples were stored frozen whole at 
20°C prior to use in the RT-PCR. All shellfish samples were
processed from whole animals.
Shellfish processing and virus extraction and purification.
The procedure for shellfish processing has been previously described in
full (12). Essentially, shellfish were shucked, homogenized,
sonicated, and centrifuged, and supernatants were precipitated with
polyethylene glycol. For seeding experiments and environmental samples,
50 g of shellfish flesh was processed. For shellfish associated
with gastroenteritis outbreaks, up to 50 g was processed
(depending on sample availability). Resuspended pellets were sonicated
and centrifuged prior to further virus purification by extraction with
1,1,2-trichloro-2,2,1-trifluoroethane (Freon TF) followed by
centrifugal concentration and storage at 20°C. Extracts at this
stage were termed purified concentrates.
Extraction of viral RNA purified concentrates.
The RNA
extraction procedure has been previously described in full
(12). Briefly, a reaction mix of glass powder matrix and
guanidine isothiocyanate (GITC) was used to extract total nucleic acid
from purified shellfish concentrates. Shellfish tissue weight
equivalents extracted were 7 g (neat), and a 1:3 dilution in PBSa
(2.3 g), for all seeding experiments and environmental samples. GITC
serves to denature cellular and nucleoprotein complexes (thereby
releasing the RNA) and protects the RNA in the sample from digestion by
nucleases. RNA bound to the glass powder was washed with GITC, ethanol,
and acetone prior to elution in Tris buffer. RNA was then precipitated
in ethanol, and RT-PCR was performed.
Oligonucleotide primers.
In the first-round SRSV RT-PCR, a
broadly reactive primer combination of the three primers, G1-G2-SM31,
was used. The sense primers, G1 and G2, were derived from published
SRSV RNA polymerase sequences and were designed to anneal specifically
with genogroup I and genogroup II strains, respectively. The antisense
primer, SM31, has previously been described (20). The
internal (nested) primers were a previously described primer pair,
NI-E3, which amplify a 113-bp region of the RNA polymerase gene
(corresponding to nucleotides 4756 to 4867 of Norwalk virus) and have
been shown to detect more than 90% of strains which were circulating
in the UK in 1993 and 1994 (6). Primer sequences and genomic
locations are given in Table 1.
Reverse transcription and RT-PCR.
SRSV RT-PCR was performed
by resuspending RNA pellets in 6.9 µl of sterile water, adding 20 U
of RNase inhibitor (RNAsin; Promega)-1 µl of 50 mM random hexamers
(PdN6; Pharmacia Biotech), and overlaying the pellets with 50 µl of
mineral oil (400-5; Sigma). The mixture was heated at 70°C for 5 min,
chilled on ice, and then added to 8.1 µl of reaction mix containing
(final concentrations) 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 5 mMgCl2, 0.2 mM (each) deoxynucleoside triphosphate (dNTP)
(Pharmacia Biotech), and 100 U of Moloney murine leukemia virus RT
(fast protein liquid chromatography pure, cloned Moloney murine
leukemia virus; Life Technologies). Reverse transcription was performed
at room temperature for 10 min followed by incubation at 37°C for
1 h. The reaction mixture was terminated by incubation at 95°C
for 5 min, the tubes were then chilled on ice, and 15 µl of RT mix
was added to 35 µl of PCR mix (10 mM Tris [pH 8.3], 50 mM KCl, 1.5 mM MgCl2, 0.2 mM [each] dNTP, 20 pmol of each primer
[G1-G2 and SM31], 1 U of Taq polymerase). After an initial
denaturation at 94°C for 2 min, 30 amplification cycles of 95°C for
1 min, 40°C for 1 min, and 72°C for 1 min were performed followed
by a final extension of 72°C for 10 min. Second-round amplification
was carried out with 1 µl of the first-round amplicon and 49 µl of
RT-PCR mix containing (final concentrations) 10 mM Tris-HCl (pH 8.8),
50 mM KCl, 1.5 mM MgCl2, 0.2 mM (each) dNTP, and 20 pmol of
each primer (NI and E3). The cycling parameters were unchanged. RT-PCR
amplicons were analyzed by electrophoresis of 20 µl of reaction mix
in agarose gels (4% Nusieve 3:1; Flowgen) at 10 V/cm for 1.5 to 2 h. First-round primers G1-G2 and SM31 amplify either a 270-bp (G2-SM31)
or a 190-bp (G1-SM31) region of the RNA polymerase gene, and nested
primer pair NI-E3 amplifies a 113-bp region. Molecular weights were
determined by comparison with a 1-kb DNA ladder (Life Technologies).
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RESULTS |
Evaluation of the specificity and sensitivity of the nested RT-PCR
assay.
A nested RT-PCR with two primer sets derived from
alignments of the polymerase region of published SRSV genomic sequences was developed. The assay was evaluated for specificity and sensitivity by using a panel of 21 fecal samples which contained SRSV particles by
electron microscopy. These SRSVs had previously been characterized by
RT-PCR and sequencing and were selected to represent the broad range of
diversity seen among genogroup I and genogroup II strains (Fig.
1). These strains were tested by both a
single-round RT-PCR with primers NI-E3 and the nested RT-PCR. The
results (Table 2) show that the
single-round RT-PCR amplified 13 of the 21 strains while the nested
RT-PCR detected 15 of the 21 strains. The nested PCR detected all but
one of the strains amplified by the single-round RT-PCR and also
amplified a further three strains, showing that the nested RT-PCR gave
wider strain cross-reactivity than did the single-round RT-PCR.
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FIG. 1.
Phylogenetic analysis of strains used for evaluation of
the nested RT-PCR assay. A panel of 21 fecal samples which had been
demonstrated to contain SRSV by electron microscopy was used in this
study. The SRSVs had previously been characterized by RT-PCR and
sequencing and were selected to reflect the broad range of diversity
seen among strains detected in the UK in recent years. The phylogenetic
analysis was performed on a 170-bp region of the RNA polymerase with
the Clustal component of the Megalign program (DNAStar). Published
sequences accessed from GenBank of Snow Mountain agent (L23831), Hawaii
virus (U07611), Melksham virus, Mexico virus (U22498), Southampton
virus (L07418), Desert Shield virus (U04469), and Norwalk virus
(M87661) are also included. Branch lengths are indicated by the
unbroken lines; dotted lines are used to provide a balanced display of
the phylogram and do not represent actual branch lengths.
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TABLE 2.
Single-round and nested RT-PCR results for a panel
of genomically representative genogroup I and II SRSVs
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The single-round and nested RT-PCRs were further compared for
sensitivity of SRSV detection. Five fecal samples containing
SRSVs
(genogroup I strains 2 and 5 and genogroup II strains 10,
15, and 18)
were titrated in PBSa dilutions from 10
1 to
10
7, and the diluted fecal extracts were tested by both
the direct
and the nested procedures (Table
3). The titer was taken as the
highest
dilution in which a specific amplicon was detected in
an ethidium
bromide-stained agarose gel. By single-round RT-PCR,
both genogroup I
strains were detectable to a dilution of only
10
1,
whereas by nested RT-PCR, the samples were positive at dilutions
of
10
4 and 10
2. All three samples containing
genogroup II strains were positive
at a dilution of 10
2
by single-round RT-PCR but could be detected at dilutions from
10
4 to 10
6 by nested RT-PCR. Thus,
significant increases in titer were demonstrated
by the nested
procedure for all five strains, demonstrating that
the nested RT-PCR
gives a higher sensitivity of detection for
both genogroup I and
genogroup II strains in fecal samples. Previous
studies (
12,
13) have shown the potent inhibitory potential
of molluscan
shellfish extracts for RT and/or
Taq polymerase enzymes
used
in the RT-PCR assay. The sensitivities of the single-round
and nested
RT-PCRs were compared for the detection of SRSV in
the presence of
shellfish meat. Commercially produced oysters
(
C. gigas)
were processed as previously described (
12) to the
purified
shellfish concentrate stage. Seeding experiments were
then performed by
adding 50 µl of SRSV (Mexico strain) fecal extract
dilution to 350 µl of either shellfish concentrate (equivalent
to 7 g of
shellfish meat) or PBSa prior to nucleic acid extraction
as previously
described (
12). Experiments were conducted with
SRSV
fecal extract dilutions (in PBSa) of 1:100, 1:500, 1:1,000,
1:5,000, 1:10,000, and 1:50,000. Sensitivities were compared for
both nested and single-round RT-PCRs (Fig.
2). A positive result
was taken as the
presence of a specific amplicon detected in an
ethidium bromide-stained
agarose gel. When SRSV extracts were
seeded into PBSa, the first round
of the nested RT-PCR (G2-SM31)
and the single-round (NI-E3) RT-PCR gave
comparable results with
amplicon bands visible to a 1:1,000 dilution of
fecal extract
(Fig.
2, gels A and B). The second round of the nested
RT-PCR
showed amplicon bands visible to a 1:10,000 dilution of fecal
extract (Fig.
2, gel C), confirming the increased sensitivity
of the
nested PCR previously observed. By contrast, when SRSV
extracts were
seeded into shellfish concentrates neither the first
round of the
nested RT-PCR (G2-SM31) nor the single-round (NI-E3)
RT-PCR gave
positive amplicon bands (Fig.
2, gels D and E). This
confirms previous
observations showing the inhibitory nature of
shellfish for RT-PCR
(
12). However, the second round of the
nested RT-PCR gave
amplicon bands at up to a 1:5,000 dilution
of SRSV fecal extract seeded
into shellfish concentrates (Fig.
2, gel F). The nested RT-PCR
therefore achieved at least a 50-fold
improvement in sensitivity over
that obtained with a single-round
PCR when used to detect SRSV in
shellfish extracts. This improvement
was only twofold less than the
optimum observed when SRSV was
seeded into PBSa rather than shellfish
extracts. This data suggests
that the nested RT-PCR not only is much
more sensitive than the
single-round RT-PCR for detecting SRSVs in
shellfish extracts
but also overcomes difficulties with the residual
RT-PCR inhibitors
left after the standard shellfish processing
procedure.
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TABLE 3.
Endpoint titration of five fecal samples containing SRSVs
and tested by the single-round and nested RT-PCRs
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FIG. 2.
Comparison of sensitivities of single-round and nested
PCRs in the presence and absence of shellfish meat. A fecal sample
containing SRSV was diluted in PBSa (gels A to C) and in purified
shellfish concentrates (gels D to F) prior to extraction and RT-PCR. In
each gel, lanes are as follows: 1, 1-kb molecular size marker; 2, 1:100
dilution of fecal sample; 3, 1:500 dilution; 4, 1:1,000 dilution; 5, 1:5,000 dilution; 6, 1:10,000 dilution; 7, 1:50,000 dilution; 8, positive control. Gels A and D show products of a single-round RT-PCR;
gels B and E show single-round products from the nested RT-PCR; gels C
and F show the nested RT-PCR second-round amplicons. Arrowheads denote
positions of first-round and second (or single)-round PCR products.
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Application of the nested RT-PCR for the detection of SRSV in field
samples.
Previous studies (14) have shown the
difficulty often experienced in detecting SRSV-specific RT-PCR
amplicons in environmental and outbreak shellfish samples. RT-PCR
amplicons were frequently visible only through the added sensitivity of
Southern blot hybridization and were of insufficient quantity for
further characterization of the detected SRSV strain(s) by sequencing.
The nested RT-PCR was further evaluated by using environmental and
outbreak shellfish samples previously found to be only weakly positive
by single-round RT-PCR in order to establish its performance with field
material. Figure 3 shows the analysis by
both single-round and nested RT-PCRs of three shellfish samples from
polluted harvesting areas and two shellfish samples associated with
food poisoning outbreaks. SRSV-specific amplicons were not visible by
gel electrophoresis in any sample following single-round RT-PCR.
However, all samples gave positive bands by nested RT-PCR. This shows
that, for all the field material tested, the nested RT-PCR was more
sensitive or less susceptible to inhibition than the single-round
RT-PCR. Moreover, nested RT-PCR amplicon bands were suitable for
further characterization.
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FIG. 3.
Application of nested PCR to field samples. Gels A to C
show RT-PCR results for shellfish samples from three contaminated
harvesting areas; gels D and E show results for samples implicated in
two outbreaks of gastroenteritis. Lanes are as follows: 1, molecular
weight markers; 2, single-round RT-PCR products; 3, nested RT-PCR
first-round products; 4, nested RT-PCR second-round products; 5, nested
RT-PCR-positive control. Arrowheads denote positions of first-round and
second (or single)-round PCR products.
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Detection of SRSVs during commercial shellfish production.
The
nested RT-PCR procedure was applied to detection of SRSVs in
commercially produced shellfish. Applications for commercial production
included evaluation of the effectiveness of the RT-PCR for detection of
virus contamination in end-product shellfish sold to the consumer and
of the effectiveness of commercial shellfish purification for removal
of SRSVs. These studies were performed with oysters (C. gigas) purified in a commercial processing plant associated with
outbreaks of infectious disease during February and March 1996. End-product monitoring of oysters from this particular plant during
this period proved valuable for demonstrating batches contaminated with
SRSVs. Figure 4 shows SRSVs detected by
RT-PCR in three of four samples tested during March 1996 but not in
samples taken during April and June. These findings were consistent
with epidemiological data showing that shellfish-associated outbreaks of infectious disease occur predominantly during the winter months (22). Further monitoring of shellfish for SRSVs before and
after purification during this period clearly showed that, although the
SRSV titers were reduced during purification (as judged by RT-PCR band
intensity), virus was not always completely cleared by this commercial
processing (Fig. 5).
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FIG. 4.
Application of nested RT-PCR for detecting SRSV in
oysters sold for consumption. Products from a commercial shellfish
supplier associated with gastroenteritis incidents were tested for
SRSVs. Lanes: 1, 1-kb molecular size marker; 2, result for shellfish
dated 8 March 1996; 3, result for shellfish dated 14 March 1996; 4, result for shellfish dated 22 March 1996; 5, result for shellfish dated
29 March 1996; 6, result for shellfish dated 26 April 1996; 7, result
for shellfish dated 7 June 1996; 8, nested RT-PCR-positive control. The
arrowhead denotes the position of the nested PCR product.
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FIG. 5.
Effect of shellfish purification on SRSV content.
Shellfish were tested by RT-PCR before and after commercial
purification. Lanes: 1, 1-kb molecular size marker; 2, batch dated 29 March 1996 before purification; 3, that batch after purification; 4, batch dated 22 February 1996 before purification; 5, that batch after
purification; 6, batch dated 14 March 1996 before purification; 7, that
batch after purification; 8, nested RT-PCR-positive control. The
arrowhead denotes the position of the nested PCR product.
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DISCUSSION |
This report describes the successful development of a nested
RT-PCR for detection of SRSVs in molluscan shellfish.
The procedure was first evaluated by using a panel of
well-characterized strains which represented the diversity of SRSVs detected in the last 9 years in the UK and consequently those that
might be anticipated to be detected in shellfish. This showed that the
nested RT-PCR assay detected a broader range of SRSVs than did the
single-round RT-PCR previously described (6). The nested
RT-PCR detected 3 of 8 genogroup I strains and 12 of 13 genogroup II
strains, and although ideally the RT-PCR would detect all strains, a
catchall primer pair has not, to date, been identified due to the
extensive genomic diversity among SRSVs. Recent data have shown
that genogroup II strains have predominated in recent years
(8), and the nested RT-PCR assay detects the vast majority
of these strains.
Comparisons of sensitivities of detection between the single-round and
nested RT-PCRs were performed with five fecal samples containing
genomically distinct SRSVs. Again, due to the genomic diversity among
SRSVs, the primer pairs used may contain mismatches with certain
strains which may decrease the sensitivity of detection for these
strains. Thus, it is necessary to determine the sensitivity of the
assay by using a range of strains. This investigation indicated that
the nested RT-PCR, as with the single-round RT-PCR, had differing sensitivities for genogroup I and genogroup II strains. However, there
was a significant increase in the sensitivity of detection (101- to 104-fold) for all strains tested.
Seeding experiments demonstrated that the nested RT-PCR was also
significantly more sensitive for SRSV detection in processed shellfish
extracts and overcame the residual PCR inhibition frequently associated
with such extracts. Application to a panel of shellfish samples from
polluted harvesting areas and associated with outbreaks of infectious
disease clearly demonstrated the value of the nested RT-PCR for
field samples. The nested RT-PCR assay again showed a greater
sensitivity and a decreased susceptibility to inhibitory substances in
shellfish meats, thus overcoming the limitations previously
experienced with a single-round RT-PCR (14).
This procedure was also applied to monitoring aspects of commercial
shellfish production. The nested RT-PCR proved capable of detecting
SRSVs in processed shellfish sold for consumption from a commercial
supplier associated with incidents of gastroenteritis due to oyster
consumption. Further monitoring showed that, although the commercial
purification routinely applied to these oysters appeared to reduce
virus content, SRSVs were not reliably eliminated. These findings
concur well with the available epidemiological evidence on oyster
contamination with SRSVs. The nested RT-PCR should prove valuable for
further studies on the behavior of SRSVs during commercial processes
such as purification and relaying. In addition, the procedure has
applications for monitoring shellfish harvesting areas at risk of
contamination with SRSVs, for investigation of SRSV contamination in
the products of shellfish producers associated with outbreaks, and for
direct investigation of shellfish causing illness. The assay may also
have potential applications in other areas of environmental monitoring,
including the detection of SRSVs in sewage, waters, and foodstuffs
other than shellfish.
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FOOTNOTES |
*
Corresponding author. Mailing address: Enteric and
Respiratory Virus Laboratory, Virus Reference Division, Central Public Health Laboratory, Colindale, London NW9 5HT, United Kingdom. Phone:
181 200 4400, ext. 3437. Fax: 181 200 1569. E-mail:
jgreen{at}phls.co.uk.
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Appl Environ Microbiol, March 1998, p. 858-863, Vol. 64, No. 3
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
