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Applied and Environmental Microbiology, November 1999, p. 4709-4714, Vol. 65, No. 11
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
A Method To Detect Low Levels of Enteric Viruses in
Contaminated Oysters
Y.-S. Carol
Shieh,1,*
Kevin R.
Calci,1 and
Ralph S.
Baric2
Gulf Coast Seafood Laboratory, U.S. Food and
Drug Administration, Dauphin Island, Alabama
36528,1 and Program in Infectious
Diseases, Department of Epidemiology, University of North Carolina,
Chapel Hill, North Carolina 275992
Received 15 April 1999/Accepted 4 August 1999
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ABSTRACT |
Direct isolation and identification of pathogenic viruses from
oysters implicated in gastroenteritis outbreaks are hampered by
inefficient methods for recovering viruses, naturally occurring PCR
inhibitors, and low levels of virus contamination. In this study we
focused on developing rapid and efficient oyster-processing procedures
that can be used for sensitive PCR detection of viruses in raw oysters.
Poliovirus type 3 (PV3) Sabin strain was used to evaluate the efficacy
of virus recovery and the removal of PCR inhibitors during
oyster-processing procedures. These procedures included elution,
polyethylene glycol precipitation, solvent extraction, and RNA
extraction. Acid adsorption-elution in which glycine buffer (pH 7.5)
was used was found to retain fewer inhibitors than direct elution in
which glycine buffer (pH 9.5) was used. RNA extraction in which a
silica gel membrane was used was more effective than single-step RNA
precipitation for removing additional nonspecific PCR inhibitors. The
final 10-µl volume of RNA concentrates obtained from 2 g of
oyster tissue (concentration factor, 200-fold) was satisfactory for
efficient reverse transcription-PCR detection of virus. The overall
detection sensitivity of our method was 1 PFU/g of oyster tissue
initially seeded with PV3. The method was utilized to investigate a
1998 gastroenteritis outbreak in California in which contaminated
oysters were the suspected disease transmission vehicle. A genogroup II
Norwalk-like virus was found in two of three recalled oyster samples
linked by tags to the harvest dates and areas associated with the
majority of cases. The method described here improves the response to
outbreaks and can be used for rapid and sensitive detection of viral
agents in outbreak-implicated oysters.
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INTRODUCTION |
Viral gastroenteritis cases
epidemiologically linked to the consumption of raw or undercooked
shellfish are probably caused by human enteric viruses. Human sewage
discharged from oyster-harvesting vessels was the probable cause
identified in one of the previous major outbreak investigations
(4, 27). Isolating and identifying etiological viral agents
in outbreak-implicated shellfish have been difficult because of low
levels of contamination, inefficient recovery during processing, and
high concentrations of natural PCR inhibitors in oyster tissues.
Development of a sensitive detection method that is also rapid and
efficient should improve public health responses to outbreaks because
such a method should allow workers to rapidly identify pathogens in
contaminated areas and shellfish. It should also facilitate
identification of appropriate viral indicators that can be used to
prevent future outbreaks.
The methods used to detect enteric viruses in shellfish consist of the
following two major elements: (i) separation and concentration of
viruses from shellfish tissue components and (ii) detection of viruses
in shellfish concentrates by molecular techniques or cell culture
infectivity assays. Molecular techniques, such as PCR, are the
preferred techniques for detection of viral pathogens that are
noncytopathic or nonculturable (e.g., Norwalk-like virus [NLV] and
hepatitis E virus). Successful PCR detection relies on effective
removal of natural inhibitors and efficient recovery of viruses from
oysters during processing. The initial step commonly used to elute
viruses from oyster tissue includes direct alkaline elution (13,
14, 16) and acid adsorption-elution (6, 23, 24).
Different eluants containing glycine, beef extract, and other compounds
have been compared to determine their virus recovery (16)
and PCR compatibility (26) characteristics. Complex
eluants, such as beef extract, have been shown to contribute additional PCR inhibitors, which decrease the sensitivity of PCR for
detection of viruses in environmental samples. The use of butanol-chloroform during extraction has been shown to improve PCR
detection (2). When the levels of viral contamination in shellfish are low, it is necessary to include a concentration step,
such as polyethylene glycol (PEG) precipitation, organic flocculation,
or ultracentrifugation. After concentration, additional steps to remove
PCR inhibitors from the processed concentrates are critical in order to
have a sensitive PCR test. RNA extraction (12, 22) and
nested PCR (10) have been found to improve significantly the
sensitivity of virus detection with environmental and clinical samples.
Attempting to avoid PCR inhibitors, workers in previous studies
concentrated limited quantities of oysters for the final PCR examination or included sophisticated and extensive processing steps to
clean up the concentrates. Processing and analysis of small samples can
result in false-negative PCR results when the level of virus is low.
Similarly, extensive processing can produce false-negative results by
decreasing recovery because of increased manipulation and handling. The
objective of this study was to develop rapid and simple processing
procedures that can be used to extract and recover enteric viruses
efficiently from 25 to 50 g of oyster tissue in a concentrated
volume (
100-fold concentration factor) for sensitive PCR detection.
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MATERIALS AND METHODS |
Cell cultures and viruses.
Human rhabdomyosarcoma (RD) cells
were grown to confluence in Eagle's minimum essential medium
containing 10% fetal bovine serum, kanamycin (250 µg/ml), and
gentamicin (50 µg/ml) in 25-cm2 flasks or 60-mm-diameter
dishes. Confluent cells were maintained in the same medium except that
the fetal bovine serum concentration was 2%. Poliovirus type 3 (PV3)
Sabin strain was grown in RD cells.
Virus recovery.
PV3 was seeded into shucked oysters and was
concentrated by the processing procedures described below. The levels
of virus recovered from processing were quantified by performing a
plaque assay, and then virus recoveries were determined. Serial 10-fold dilutions of each processed concentrate were inoculated onto confluent RD cells in 60-mm-diameter dishes. Cytopathology resulting from viral
replication started to appear 36 to 48 h after inoculation, and
viral plaques were enumerated 72 h after inoculation.
Oysters and oyster processing.
Commercial-size eastern
oysters (Crassostrea virginica) that were 3.5 to 5 in. long
were purchased or collected from Mobile Bay in Alabama and Apalachicola
Bay in Florida between May 1997 and March 1998. For low-level
virus-seeding studies, oysters were depurated with UV-treated seawater
for 1 to 2 weeks prior to processing and seeding. Precise levels of
viruses in oysters were obtained by inoculating a PV3 stock preparation
directly into freshly shucked oysters. After 10 to 15 min of
incubation, the seeded oysters were processed by using the procedures
described below.
Approximately 3 weeks after harvesting, three samples of recalled
outbreak-implicated oysters (Crassostrea gigas) from
California were shipped in a chilled, insulated container by overnight
express to the Food and Drug Administration (FDA) Gulf Coast Seafood
Laboratory. Immediately after arrival, the oysters were examined, and
viable oysters were shucked (without liquor and adductors). Two to four oysters from each sample (weight, slightly more than 25 g) were stored in each sterile container and were frozen at 
70°C.
The oyster-processing procedure consisted of the following steps: step
1, homogenization of 25 g of oyster tissue in 175 ml
of cold
sterile deionized water; step 2, acid adsorption of viruses
to oyster
solids from the homogenates by adjusting the pH 5.0
after addition of
water to reduce the conductivity to less than
2,000 µS
(
23) (pellets were collected after centrifugation at
2,000 ×
g for 20 min); step 3, elution of viruses with
175 ml
of 0.05 M glycine-0.15 M NaCl (pH 7.5) (the mixture was shaken
for 15 min at room temperature, and the supernatant was collected
after
centrifugation at 5,000 ×
g for 20 min at 4°C); step
4,
precipitation of viruses by using 8% PEG 8000-0.3 M NaCl at 4°C
for 4 h (pellets were collected after centrifugation at
6,700
×
g for 30 min, and pellets were suspended in 10 ml of phosphate-buffered
saline); step 5, solvent extraction of viruses
with an equal volume
of 1,1,2-trichloro-1,1,2-trifluoroethane (Freon)
(the supernatant
was collected after centrifugation at 1,700 ×
g for 30 min); step
6, precipitation of viruses again by using
8% PEG 8000-0.3 M NaCl
at 4°C for 4 h (pellets were collected
after centrifugation at
14,000 ×
g for 15 min); and
step 7, RNA extraction and purification
of the PEG precipitate by using
a silica gel membrane (Qiagen
Inc., Valencia, Calif.). A 25-g portion
of oyster tissue was concentrated
approximately 150-fold to obtain a
final RNA volume of 160 µl.
During the development of the method,
acid adsorption-elution
(steps 1 through 3) was compared to direct
alkaline elution of
oysters homogenized with 10 volumes of 10%
tryptose phosphate
broth-0.05 M glycine (pH 9.5) (
13,
14).
RNA extraction and
purification with silica gel (step 7) were compared
with two other
RNA extraction procedures, single-step RNA extraction
(
5) alone
and single-step RNA extraction combined with
Sephadex spin column
chromatography (
19,
22,
25).
RT and PCR.
Reverse transcription (RT) of poliovirus genomic
RNA was carried out with a panenterovirus antisense primer
(7) at 42°C for 1 h immediately after the RNA was
denatured at 98°C for 5 min. Panenterovirus PCR amplification was
performed for 25 cycles, with each cycle consisting of 95°C for 1.5 min, 55°C for 1 min, and 72°C for 1.5 min, which yielded an
amplified 196-bp product. The panenterovirus primer and probe sequences
were selected from the highly conserved 5' nontranslated region and
have been described previously (20). The primers used
amplify sequences of human enteroviruses, but they did not amplify
sequences of several animal enteroviruses tested.
Polymerase regions of NLV genogroup I (G1) and genogroup II (G2) were
examined with NLV primers and probes, as described by
Ando et al.
(
1). RT of NLV was carried out in a 30-µl reaction
mixture
at 42°C for 1 h with consensus primer SR33 for G1 and
G2. PCR
amplification of G2 was followed by addition of sense
primer SR46.
Amplification of G1 was carried out with a mixture
of three sense
primers, SR48, SR50, and SR52. All NLV PCR were
performed for 40 cycles, with each cycle consisting of 94°C for
1 min, 50°C for 1 min, and 68°C for 2 min (shorter annealing time
and higher extension
temperature than Ando et al. [
1]
used).
Analysis of DNA by gel electrophoresis and hybridization.
The 196-bp PCR product for panenterovirus and the 123-bp PCR product
for NLV were analyzed by 1.8% agarose gel electrophoresis, revealed by
ethidium bromide (EtBr) staining, and Southern transferred to
positively charged nylon membranes (Roche Molecular Biochemicals, Indianapolis, Ind.) for oligohybridization. The membranes were prehybridized for 2 to 4 h at 52°C for enterovirus and at 58°C for NLV and were then hybridized with digoxigenin-labelled probes at
the same temperature. The hybridization and colormetric detection conditions recommended by the manufacturer (Roche Molecular
Biochemicals) were used.
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RESULTS |
Recovery of virus during oyster processing.
Several processing
steps, including virus elution, PEG precipitations, solvent extraction,
and RNA extraction, were used, modified, and evaluated in this study in
order to effectively remove PCR inhibitors and to efficiently recover
viruses from oysters. Virus recoveries were carried out and measured by
seeding PV3 Sabin strain (103 to 105 PFU/g of
oyster) into oysters, concentrating the PV3 by using the steps
described above, and then assaying the sample concentrates with RD
cells. For the first step, acid adsorption-elution and direct alkaline
elution were compared. A lower percentage of the PV3 (59%) was
recovered by acid adsorption-elution than by direct alkaline elution
(Table 1); this finding may be attributed
to the two centrifugation steps used during acid adsorption-elution rather than the one centrifugation step used during direct alkaline elution. The final recoveries of viruses were not significantly different (10.3 and 10.5%) (Table 1). The viruses in oysters were
concentrated more than 100-fold, and approximately 1 log of the seeded
PV3 was lost.
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TABLE 1.
Poliovirus recoveries from seeded oysters processed by
our method, beginning with acid adsorption-elution or direct
alkaline elution
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Oysters were frequently frozen prior to analysis in order to minimize
degradation of the viral RNA. The effect of freeze-thawing
on the
oyster tissue matrix, which might have resulted in poor
viral
adsorption, was investigated in trial 3 (Table
1). PV3
was seeded into
fresh oysters, as well as into oysters that were
frozen and thawed
three times prior to seeding. The recoveries
of PEG-precipitated PV3
from the two kinds of oysters were not
significantly different (35%
for fresh oysters [Table
1] and
39% for freeze-thawed oysters after
the first PEG precipitation,
and 14% for fresh oysters [Table
1] and
16% for freeze-thawed
oysters after the second PEG
precipitation).
Removal of RT-PCR inhibitors from oysters.
The final RNA
concentrates obtained in trials 1 and 2 (Table 1) were compared in
order to study RT-PCR interference. The RNA concentrates derived from
the process by acid adsorption-elution produced slightly stronger
RT-PCR signals than comparable RNA concentrates derived from the
process by direct alkaline elution produced (Fig.
1), even when the virus recoveries for
the processes were similar (Table 1). Stronger PCR signals may have
been due to efficient removal of inhibitors that resulted from the
first centrifugation which removed the supernatant and the second
centrifugation which removed the oyster solids. Acid adsorption-elution
was selected for the final method in this study, because efficient
removal of inhibitors resulted in sensitive detection of viruses in
final RNA concentrates.
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FIG. 1.
RT-PCR-amplified PV3 in oyster concentrates processed by
our method, beginning with either acid adsorption elution or direct
alkaline elution. Lanes 1 through 7, trial 1 performed with direct
alkaline elution (lanes 1 through 3) and acid adsorption-elution (lanes
4 through 6) (lanes 1 and 4, amplified PV3 in 0.025 g of seeded
oysters; lanes 2 and 5, PV3 in 0.25 g of seeded oysters; lanes 3 and 6, PV3 in 2.5 g of seeded oysters; lane 7, RT-PCR negative
reagent control); lanes 8 through 12, trial 2 performed with direct
alkaline elution (lanes 8 and 9) and acid adsorption-elution (lanes 10 and 11) (lanes 8 and 10, amplified PV3 in 0.05 g of oysters; lanes
9 and 11, PV3 in 0.5 g of oysters; lane 12, RT-PCR negative
reagent control).
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RNA extraction was also evaluated and optimized to efficiently remove
RT-PCR inhibitors. The following RNA extraction procedures
were
evaluated: (i) single-step RNA extraction (procedure 1),
(ii)
single-step RNA extraction combined with Sephadex G-150 column
chromatography (procedure 2), and (iii) RNA adsorption and elution
with
a silica gel membrane (procedure 3). The efficiency of removing
inhibitors from oysters by using the three RNA extraction procedures
was evaluated by monitoring the inhibition of
Taq DNA
polymerase.
Thus, in PCR mixtures, equal quantities of PV3 cDNA were
mixed
with each of the different oyster RNA extracts. Any reduction
in
the PV3 PCR signal indicated that there were residual inhibitors
that
were not removed by extraction from the second PEG precipitates.
Single-step RNA extraction (procedure 1) did not effectively remove
inhibitors from the second PEG precipitates obtained from 3 g
of
oyster tissue (Fig.
2a,
lane 2). RNA-preferential precipitation
at pH 4.0 did not improve
removal of inhibitors (lane 3) compared
with the first precipitation at
pH 5.2 (lane 2). When Sephadex
G-150 column chromatography was added
(procedure 2), however,
PCR amplification of PV3 cDNA was improved, as
shown in Fig.
2a,
lanes 5 through 7. The inhibitors that originally
remained in
3 g of oyster extract (Fig.
2a, lane 8) were removed
by Sephadex
column chromatography combined with single-step RNA
precipitation
(lane 6). The amplified signals were restored in inverse
proportion
to the quantity of oyster concentrate incorporated into each
PCR
mixture (Fig.
2a, lanes 5 through 7).
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FIG. 2.
Comparison of RT-PCR amplification of PV3 that were
mixed with oyster extracts obtained with three RNA extraction
procedures: PCR amplification of PV3 cDNA mixed with oyster extracts
obtained with procedures 1 and 2 (a) and RT-PCR amplification of PV3
RNA with and without oyster extracts obtained with procedure 3 (b). We
examined amplified PCR products in EtBr-stained gels (top panels) and
the corresponding Southern blots hybridized with an inner oligomer
labelled with digoxigenin (bottom panels). (a) PCR amplification of PV3
cDNA with oyster RNA extracts obtained with procedure 1 (lanes 2, 3, and 8) and PCR amplification of PV3 cDNA with extracts obtained with
procedure 2 (lanes 5 through 7). Lane 1, positive control containing 10 µl of PV3 cDNA; lane 2, 10 µl of PV3 cDNA and 3 g of oyster
RNA precipitated at pH 5.2 by procedure 1; lane 3, 10 µl of PV3 cDNA
and 3 g of oyster RNA precipitated at pH 4.0 by procedure 1; lane
4, blank; lane 5, 5 µl of PV3 cDNA and 6 g of oyster extract
obtained with procedure 2; lane 6, 5 µl of PV3 cDNA and 3 g of oyster extract obtained with procedure 2;
lane 7, 5 µl of PV3 cDNA and 0.625 g of oyster extract obtained with
procedure 2; lane 8, 5 µl of PV3 cDNA and 3 g of oyster RNA
precipitated at pH 5.2 by procedure 1; lane 9, PCR reagent negative
control; lane 10, DNA molecular weight standard. (b) RT-PCR
amplification of PV3 RNA with and without oyster RNA extracts obtained
with procedure 3. Lanes 1 through 4, RT-PCR amplification of PV3 in
phosphate-buffered saline (lane 1, 20 PFU; lane 2, 2 PFU; lane 3, 0.2 PFU; lane 4, 0.02 PFU); lane 5, PCR reagent negative control; lane 6, oyster extract negative control; lanes 7 through 10, amplification of
PV3 with 1.2 g of oyster extract per reaction mixture (lane 7, 20 PFU; lane 8, 2 PFU; lane 9, 0.2 PFU; lane 10, 0.02 PFU); lanes 11 through 14, amplification of PV3 with 2.1 g of oyster extract per
reaction mixture (lane 11, 20 PFU; lane 12, 2 PFU; lane 13, 0.2 PFU;
lane 14, 0.02 PFU); lane 15, DNA molecular weight standard.
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RNA extraction procedure 3, in which a silica gel membrane was used,
was investigated because Sephadex column packing (procedure
2) is
complex and can result in inconsistent elution volumes.
The total
processing time required for procedure 3, in which RNA
was adsorbed to
and eluted from a silica gel membrane, was less
than 1 h. PV3
stock solutions (20, 2, 0.2, and 0.02 PFU) with
and without RNA
extracts prepared from February Gulf Coast oysters
by using procedure 3 were compared for RT-PCR amplification. RNA
extracts derived from
1 g (Fig.
2b, lanes 7 through 10) and 2
g (lanes 11 through
14) of oysters were incorporated into each
reaction mixture, and we
found that these extracts did not interfere
with RT-PCR amplification,
as shown by the similar detection limits.
Up to 3 g of oyster
tissue could be extracted and incorporated
into each PCR mixture
without major interference. The RNAs derived
from silica gel membranes
were found to be more consistent and
reliable for successful PCR
amplification than the RNAs obtained
from Sephadex column
chromatography and single-step RNA extraction.
Therefore, RNA
extraction with a silica gel membrane (procedure
3) was selected due to
its rapid, simple, and reliable removal
of
inhibitors.
Overall detection sensitivity as determined with low levels of
virus seeding.
The overall detection sensitivity of our method was
determined by inoculating the PV3 Sabin strain into 25 g of
freshly shucked oysters, processing the seeded oysters, and then
examining final RNA concentrates by RT-PCR. Experiments were conducted
with low initial PV3 seeding densities (280, 58, 5, 1.2, and 0.2 PFU/g of oyster tissue). As shown in Table 2,
the PV3 detection limit was established through trial D, in which
oysters were seeded with 1.2 PFU of PV3/g. A 10-µl volume of trial D
RNA concentrate containing 1.6 PFU was PCR positive, whereas 2 µl of
an RNA concentrate containing 0.3 PFU was PCR negative (Table 2 and
Fig. 3). The trial D PCR results were
consistent with the results of all other trials, especially trial C, in
which positive PCR results were obtained with 2 µl of RNA concentrate
containing 1.2 PFU in 0.24 g of oyster tissue (Table 2). Overall,
the level of virus detectable by the method was 1.2 PFU/g of initially
seeded oysters (trial D) or 1.2 PFU of PV3 per PCR mixture (trial C).
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FIG. 3.
RT-PCR amplification of low levels of PV3 in seeded
oysters. Lane 1, DNA molecular weight standard; lanes 2 and 3, amplified PV3 in oysters seeded with 58 PFU/g (trial B) (lane 2, 10 µl of RNA concentrate per PCR mixture; lane 3, 2 µl of RNA
concentrate per PCR mixture); lanes 4 and 5, amplified PV3 in oysters
seeded with 5 PFU/g (trial C) (lane 4, 10 µl of RNA concentrate per
PCR mixture; lane 5, 2 µl of RNA concentrate per PCR mixture); lanes
6 and 7, amplified PV3 in oysters seeded with 1.2 PFU/g (trial D) (lane
6, 10 µl of RNA concentrate per PCR mixture; lane 7, 2 µl of RNA
concentrate per PCR mixture); lane 8, control containing 10 µl of RNA
concentrate (trial F); lane 9, RT-PCR reagent negative control.
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Virus detection in outbreak-implicated oysters.
The method
described above was applied to oysters implicated in a gastroenteritis
outbreak consisting of 171 cases that occurred in 44 clusters located
in seven counties of California (3). Nausea, vomiting,
diarrhea, chills, stomach cramps, and low-grade fevers were common
symptoms among the patients who consumed raw or undercooked oysters
harvested from Tomales Bay in California. As traced by the
oyster-tagging system, illness-associated oysters were harvested by
certified growers from approved areas in the mid to outer bay starting
on 29 April 1998 (3). The harvested oysters (unshucked) were
presumably stored under conditions set by National Shellfish Sanitation
Program for human consumption. Three recalled oyster samples, selected
on the basis of their harvest dates and proximity to the majority of
cases, were shipped to the FDA Gulf Coast Seafood Laboratory for viral
pathogen analysis. A 25-g aliquot of each shucked oyster tissue was
processed individually, and final RNA concentrates were examined by
RT-PCR for the presence of enterovirus, as well as NLV G1 and G2. NLV
G2 was found in two samples, samples 87 and 90, by using RT-PCR and
Southern hybridization (Table 3). When
panenterovirus PCR primers were used, we found enterovirus in sample
88. The NLV G2 in samples 87 and 90 was characterized, and the identity
was confirmed by nucleotide sequencing (data not shown). The fact that
all three oyster samples contained human enteric viruses indicated that
the oysters were contaminated with human wastes.
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DISCUSSION |
In the United States, identification of viral etiological agents
in outbreak-implicated shellfish has not been successful except in rare
instances (8, 15). The lack of efficient methods for
recovering viruses and removing inhibitors from shellfish hinders
sensitive PCR detection of viruses, especially in shellfish contaminated with low levels of viruses. In this study we improved the
processing procedures by streamlining the steps required to obtain
reasonable virus recovery and effective inhibitor removal for the final
150- to 200-fold concentrates used. Acid adsorption-elution was found
to have a greater ability to remove inhibitors, possibly due to the
two-step process used, although direct alkaline elution with glycine
buffer (pH 9 to 10) is quick and commonly used by many researchers
(11, 13, 14, 16, 17). In addition, smaller pellets of second
PEG precipitates were observed after processing with the acid
adsorption-elution procedure. The smaller pellets were dissolved easily
by RNA buffer during the final step of RNA extraction.
The final RNA extraction step is indispensable for virus detection in
environmental samples as it effectively removes inhibitors and
extensively concentrates the final volume (22). Numerous RNA
extraction procedures have been described, and some of them have been
applied to oyster concentrates. These procedures include the use of
phenol-chloroform extraction followed by cetyltrimethylammonium bromide
purification (2, 15) and the use of glass powder (13,
14). In our study, a rapid single-step RNA extraction procedure
failed to remove inhibitors from the second oyster PEG precipitates.
When Sephadex column chromatography preceded single-step RNA
extraction, RT-PCR amplification of viruses in processed concentrates was restored. However, this two-step RNA processing (procedure 2) was
time-consuming and complex. A third procedure, in which silica gel was
used to adsorb and elute RNA, proved to be timely, required less than
1 h, and produced quality RNA more consistently and reliably. This
finding is similar to the finding of a study in which the authors
showed that guanidinium-silica gel extraction was superior to
chromatography combined with RNA precipitation, and also to
phenol-chloroform extraction combined with cetyltrimethylammonium bromide purification for detecting virus in fecal specimens
(12). In our study, the RNA extraction step did not
discriminate between viral RNAs and residual oyster RNAs. Residual
oyster RNAs did not interfere with RT-PCR, but they masked and hindered
the observation of small amplified products in EtBr-stained gels (Fig.
2). The use of Southern hybridization or the use of Microcon 100 (Amicon Inc., Beverly, Mass.) for product purification before gel
electrophoresis resolved the problem. Southern hybridization is
strongly recommended for detecting virus in environmental samples
because of its high level of sensitivity (it is approximately 1-log
more sensitive than EtBr-stained gel electrophoresis) and specificity
for recognizing specific amplified targets by nucleic acid hybridization.
The virus recoveries obtained with the method developed in this study
(Table 1) were measured by using a cell culture infectivity assay
during PV3 seeding experiments (105 to 103 PFU
of PV3/g). Since low levels of PV3 in oysters frequently did not
produce enough plaque counts on a 60-mm-diameter dish, the efficient
way to detect low levels of PV3 was to use RT-PCR, not the cell culture
infectivity assay. Using the RT-PCR assay in five low-level PV3 seeding
experiments (virus levels, 102 to 10
1 PFU/g)
(Table 2), we concluded that the overall limit of virus detection in
oysters by the method was 1.2 PFU/g of oysters initially seeded with
PV3. Because oysters were depurated first and controls did not contain
detectable levels of enterovirus (trial F), we believe that the seeded
1.2 PFU/g represented the actual virus level in the oysters. Background
enterovirus was not present in the control oysters simultaneously
examined during the January and February trials (data not shown). As
the ratio of virus particles to infectivity was greater than one, the
method could detect 1 PFU/g of oyster tissue, even when there was a
total loss of approximately 1 log of virus during processing. The
sensitivity of detection in this study (1 PFU/g) was determined by
using single-round PCR with oysters seeded initially and processed by
using all of the steps; our method is considered sensitive and
comprehensive compared to previously described methods. For example, a
sensitivity of 1 PFU of PV1 was reported for a partial processing
procedure from nucleic acid extraction to RT-PCR (13). Our
low detection limit may be attributed to enhanced removal of inhibitors
from the final 10-µl volume of RNA concentrates obtained from 1.5 to
2 g of oyster tissue. Twenty-five to 50 g of oyster tissue
can be processed and the final RNA concentrates can be obtained within
10 to 12 h.
As oyster biochemistry differs in different seasonal environments, the
inhibitor levels may vary. A previous study showed that the levels of
PCR inhibitors in oysters collected from polluted waters were different
from the levels in oysters that have been depurated (13). To
ensure that oyster biochemical variables were taken into consideration,
our method was tested periodically with oysters collected in different
months and seasons from Gulf Coast waters. In particular, oysters
harvested from January to March were utilized in five trials with low
levels of virus seeding (Table 2). During cold months, oysters may
accumulate more inhibitory substances, perhaps due to increased storage
of glycogen (9). If not removed properly by processing, the
inhibitors may alter the virus detection limit.
The method which we developed was successfully used to examine NLV in
illness-associated Tomales Bay oysters. Environmental and clinical
specimens (oysters and patient stools) were processed and examined
independently by workers in different laboratories. We found 100%
identity in 175 nucleotide sequences in the capsid gene of NLV in
oysters and a patient when NLV capsid primers were utilized
(21). During our examination of an NLV strain in implicated oysters, depurated control oysters were always negative for NLV, which
indicated that no contaminant was introduced by the processing and
examination procedures. Using 1.4 to 1.5 g of oyster tissue, we
consistently detected NLV G2 amplicons in concentrated 10-µl volumes
of RNAs obtained from two concentrates, samples 87 and 90. Perhaps
because of low levels of virus contamination, NLV G2 signals in 2-µl
portions of RNA sample concentrates were not observed consistently. It
was unlikely that a false-negative NLV result occurred in sample 88 (due to remaining inhibitors), because enterovirus in the same sample
was well amplified.
For decades (even in the 1980s and early 1990s), the etiology of the
majority of illnesses associated with shellfish consumption was unknown
(18). In recent years, NLV etiological agents have been
found mostly in patient stool samples but not in illness-implicated shellfish. The ability to find low levels of enteric viruses in shellfish allows more accurate assessment of shellfish as disease transmission vehicles. The method which we developed should improve the
public health response to viral illnesses associated with oyster
consumption by permitting rapid isolation and sensitive identification
of viral agents in oysters implicated in illnesses.
| |
ACKNOWLEDGMENTS |
This research was supported by the FDA and the U.S. Environmental
Protection Agency through the Gulf of Mexico Program (EPA IAG
identification no. DW75945791-01-0).
We thank D. W. Cook, R. M. McPhearson, G. P. Hoskin, and
P. S. Schwartz of the FDA Office of Seafood for valuable critiques during preparation of the manuscript. We thank California Department of
Health Services and FDA shellfish specialists in the Pacific region for
providing the recalled oyster samples and illness reports. The
assistance of F. A. Bencsath, W. Burkhardt III, and J. L. Mullendore in making this publication possible is also appreciated.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Gulf Coast
Seafood Laboratory, U.S. Food and Drug Administration, P.O. Box 158, Dauphin Island, AL 36528. Phone: (334) 690-3407. Fax: (334) 694-4477. E-mail: ycs{at}vm.cfsan.fda.gov.
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Applied and Environmental Microbiology, November 1999, p. 4709-4714, Vol. 65, No. 11
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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Top
Abstract
Introduction
