Membrane Stabilization and Detoxification of Acetaminophen-Mediated Oxidative Onslaughts in the Kidneys of Wistar Rats by Standardized Fraction of Zea mays L. (Poaceae), Stigma maydis.

This study evaluated membrane stabilization and detoxification potential of ethyl acetate fraction of Zea mays L., Stigma maydis in acetaminophen-induced oxidative onslaughts in the kidneys of Wistar rats. Nephrotoxic rats were orally pre- and posttreated with the fraction and vitamin C for 14 days. Kidney function, antioxidative and histological analyses were thereafter evaluated. The acetaminophen-mediated significant elevations in the serum concentrations of creatinine, urea, uric acid, sodium, potassium, and tissue levels of oxidized glutathione, protein-oxidized products, lipid peroxidized products, and fragmented DNA were dose-dependently assuaged in the fraction-treated animals. The fraction also markedly improved creatinine clearance rate, glutathione, and calcium concentrations as well as activities of superoxide dismutase, catalase, glutathione reductase, and glutathione peroxidase in the nephrotoxic rats. These improvements may be attributed to the antioxidative and membrane stabilization activities of the fraction. The observed effects compared favorably with that of vitamin C and are informative of the fraction's ability to prevent progression of renal pathological conditions and preserve kidney functions as evidently supported by the histological analysis. Although the effects were prominently exhibited in the fraction-pretreated groups, the overall data from the present findings suggest that the fraction could prevent or extenuate acetaminophen-mediated oxidative renal damage via fortification of antioxidant defense mechanisms.

Kinetics of α-amylase and α-glucosidase inhibitory potential of Zea mays Linnaeus (Poaceae), Stigma maydis aqueous extract: An in vitro assessment.

ETHNOPHARMACOLOGICAL RELEVANCE: Corn silk (Zea mays L., Stigma maydis) is an important herb used traditionally in many parts of the world to treat array of diseases including diabetes mellitus. Inhibitors of α-amylase and α-glucosidase offer an effective strategy to modulate levels of post prandial hyperglycaemia via control of starch metabolism. AIM OF THE STUDY: This study evaluated α-amylase and α-glucosidase inhibitory potentials of corn silk aqueous extract. Active principles and antioxidant attributes of the extract were also analysed. MATERIALS AND METHODS: The α-amylase inhibitory potential of the extract was investigated by reacting its different concentrations with α-amylase and starch solution, while α-glucosidase inhibition was determined by pre-incubating α-glucosidase with different concentrations of the extract followed by addition of p-nitrophenylglucopyranoside. The mode(s) of inhibition of the enzymes were determined using Lineweaver-Burke plot. RESULTS: In vitro analysis of the extract showed that it exhibited potent and moderate inhibitory potential against α-amylase and α-glucosidase, respectively. The inhibition was concentration-dependent with respective half-maximal inhibitory concentration (IC50) values of 5.89 and 0.93mg/mL. Phytochemical analyses revealed the presence of alkaloids, flavonoids, phenols, saponins, tannins and phytosterols as probable inhibitory constituents. Furthermore, the extract remarkably scavenges reactive oxygen species like DPPH and nitric oxide radicals, elicited good reducing power and a significant metal chelating attributes. CONCLUSION: Overall, the non-competitive and uncompetitive mechanism of action of corn silk extract is due to its inhibitory effects on α-amylase and α-glucosidase, respectively. Consequently, this will reduce the rate of starch hydrolysis, enhance palliated glucose levels, and thus, lending credence to hypoglycaemic candidature of corn silk.

Comparison of the effects of dietary plant sterol and stanol esters on lipid metabolism.

BACKGROUND AND AIM: To compare the cholesterol-lowering efficacy and other metabolic effects of plant sterol and stanol esters, both of which are commonly used in the dietary management of hypercholesterolaemia. METHODS AND RESULTS: The cholesterol-lowering efficacy of equivalent intakes of sterol and stanol esters and of different intakes of stanol esters were compared at 1 and 2 months, both in normal subjects and treated patients with familial hypercholesterolaemia. Systemic effects were assessed by measuring serum levels of plant sterols and of lathosterol and 7alpha-hydroxy-cholestenone, indices of sterol absorption and of cholesterol and bile acid synthesis respectively. There were no significant differences during the study between 1.6g daily of sterol and stanol esters in reducing total cholesterol (by 3-7%) or low density lipoprotein cholesterol (by 4-8%), nor between 1.6 and 2.6 g daily of stanol. However, the cholesterol-lowering effect of plant sterol esters was attenuated between 1 and 2 months. This was accompanied by increased serum plant sterols and decreased levels of 7alpha-hydroxy-cholestenone, especially in statin-treated hypercholesterolaemic patients not taking bile acid sequestrants. CONCLUSIONS: These findings suggest that absorption of dietary plant sterols suppressed bile acid synthesis, thereby diminishing their cholesterol-lowering efficacy. In contrast, plant stanols reduced plant sterol absorption and maintained their cholesterol-lowering efficacy.

Effect of a statin on hepatic apolipoprotein B-100 secretion and plasma campesterol levels in the metabolic syndrome.

OBJECTIVE: We aimed to study the effect of atorvastatin, a statin, on cholesterol synthesis and absorption and VLDL-apoB metabolism in obese men with the metabolic syndrome. METHODS: A total of 25 dyslipidaemic obese men were randomized to atorvastatin (n=13) (40 mg/day) or matching placebo (n=12) for 6 weeks. Hepatic secretion and fractional catabolic rate (FCR) of VLDL-apoB was measured using an intravenous bolus of d(3)-leucine before and after treatment. ApoB isotopic enrichment was measured using GCMS and multicompartmental modelling. Plasma lathosterol: cholesterol and campesterol:cholesterol ratios were determined to assess cholesterol synthesis and cholesterol absorption, respectively. RESULTS: Compared with placebo, atorvastatin significantly decreased (P<0.05) total cholesterol, triglyceride, LDL-cholesterol and VLDL-apoB. Plasma lathosterol:cholesterol ratio decreased from 26.4+/-2.4 to 8.8+/-0.8, while the campesterol:cholesterol ratio increased from 26.5+/-4.4 to 38.6+/-5.8 (P<0.01). Atorvastatin also increased VLDL-apoB FCR from 3.82+/-0.33 to 6.30+/-0.75 pools/day (P<0.01), but did not significantly alter VLDL-apoB secretion (12.8+/-1.7 to 13.8+/-2.0 mg/kg/day). CONCLUSIONS: In obesity, atorvastatin inhibits cholesterogenesis but increases intestinal cholesterol absorption. The increased cholesterol absorption may counteract the inhibitory effect on hepatic VLDL-apoB secretion, but it does not apparently influence enhanced catabolism of VLDL-apoB.

Treatment with atorvastatin alters the ratio of interleukin-12/interleukin-10 gene expression [corrected].

BACKGROUND: Statins have been shown to have pleiotropic effects extending beyond their ability to lower cholesterol. MATERIAL AND METHODS: Seventeen patients with heterozygous familial hypercholesterolaemia participated in a single-blind placebo controlled study. The patients underwent three treatment regimens: placebo (4 weeks), atorvastatin 10 mg day(-1) (4 weeks) and atorvastatin 40 mg day(-1) (12 weeks). Following each treatment period, serum lipids and plasma mevalonic acid were measured, mononuclear leukocytes were isolated and total RNA was prepared. The content of mRNA for IL-12p35 and IL-10 was assayed, blinded, by real-time quantitative polymerase chain reactions. RESULTS: Treatment of the subjects with atorvastatin decreased the abundance of IL-12p35 mRNA in mononuclear cells, but did not alter that of IL-10, so that the ratio of the IL-12p35 to IL-10 mRNA content was significantly reduced (P < 0.0026). The IL-12p35/IL-10 ratio correlated significantly with plasma mevalonic acid concentrations but not with serum LDL concentrations. CONCLUSIONS: This study provides evidence that atorvastatin exerts an immunomodulatory effect in vivo, characterized by a decrease in the ratio of IL-12 mRNA to IL-10 mRNA in leukocytes. The immunomodulatory effect of statins, in addition to their cholesterol-lowering properties, may contribute to the rapid cardiovascular benefit observed during treatment with statins and reduced the rate of rejection in patients with solid organ transplantation.

Determinants of variable response to statin treatment in patients with refractory familial hypercholesterolemia.

Interindividual variability in low density lipoprotein (LDL) cholesterol (LDL-C) response during treatment with statins is well documented but poorly understood. To investigate potential metabolic and genetic determinants of statin responsiveness, 19 patients with refractory heterozygous familial hypercholesterolemia were sequentially treated with placebo, atorvastatin (10 mg/d), bile acid sequestrant, and the 2 combined, each for 4 weeks. Levels of LDL-C, mevalonic acid (MVA), 7-alpha-OH-4-cholesten-3-one, and leukocyte LDL receptor and hydroxymethylglutaryl coenzyme A reductase mRNA were determined after each treatment period. Atorvastatin (10 mg/d) reduced LDL-C by an overall mean of 32.5%. Above-average responders (LDL-C -39.5%) had higher basal MVA levels (34.4+/-6.1 micromol/L) than did below-average responders (LDL-C -23.6%, P<0.02; basal MVA 26.3+/-6.1 micromol/L, P<0.01). Fewer good responders compared with the poor responders had an apolipoprotein E4 allele (3 of 11 versus 6 of 8, respectively; P<0.05). There were no baseline differences between them in 7-alpha-OH-4-cholesten-3-one, hydroxymethylglutaryl coenzyme A reductase mRNA, or LDL receptor mRNA, but the latter increased in the good responders on combination therapy (P<0.05). Severe mutations were not more common in poor than in good responders. We conclude that poor responders to statins have a low basal rate of cholesterol synthesis that may be secondary to a genetically determined increase in cholesterol absorption, possibly mediated by apolipoprotein E4. If so, statin responsiveness could be enhanced by reducing dietary cholesterol intake or inhibiting absorption.

Müllerian Inhibiting Substance lowers testosterone in luteinizing hormone-stimulated rodents.

Müllerian Inhibiting Substance (MIS) expression is inversely proportional to the serum concentration of testosterone in males after birth and in vitro studies have shown that MIS can lower testosterone production by Leydig cells. Also, mice overexpressing MIS exhibited Leydig cell hypoplasia and lower levels of serum testosterone, but it is not clear whether this is a result of MIS affecting the development of Leydig cells or their capacity to produce testosterone. To examine the hypothesis that MIS treatment will result in decreased testosterone production by mature Leydig cells in vivo, we treated luteinizing hormone (LH)-stimulated adult male rats and mice with MIS and demonstrated that it can lead to a several-fold reduction in testosterone in serum and in testicular extracts. There was also a slight decrease in 17-OH-progesterone compared to the more significant decrease in testosterone, suggesting that MIS might be regulating the lyase activity of cytochrome P450c17 hydroxylase/lyase (Cyp17), but not its hydroxylase activity. Northern analysis showed that, in both MIS-treated rats and mice, the mRNA for Cyp17, which catalyzes the committed step in androgen synthesis, was down-regulated. In rats, the mRNA for cytochrome P450 side-chain cleavage (P450scc) was also down-regulated by MIS. This was not observed in mice, indicating that there might be species-specific regulation by MIS of the enzymes involved in the testosterone biosynthetic pathway. Our results show that MIS can be used in vivo to lower testosterone production by mature rodent Leydig cells and suggest that MIS-mediated down-regulation of the expression of Cyp17, and perhaps P450scc, contributes to that effect.

Development of a reverse transcription-PCR-DNA enzyme immunoassay for detection of "Norwalk-like" viruses and hepatitis A virus in stool and shellfish.

Outbreaks of food- and waterborne gastroenteritis are being increasingly reported throughout the world. The analysis of environmental samples by newer diagnostic techniques such as reverse transcription-PCR (RT-PCR) amplification of nucleic acid has begun to identify human enteric viruses (predominantly "Norwalk-like" viruses [NLVs]) as the cause of many of these outbreaks. To streamline NLV detection from environmental samples such as shellfish, we have developed an RT-PCR-oligoprobe amplification and detection method using several new procedures that enable confirmed RT-PCR amplification and product detection in 1 day. The new steps include replacing reverse transcriptase and Taq polymerase with rTth polymerase, a heat-stable enzyme that functions as both a reverse transcriptase and DNA polymerase, in a single-tube, single-buffer, elevated temperature reaction. An internal standard Norwalk virus (NV) RNA control is added to each RT-PCR to identify sample inhibition, and thermolabile uracil N-glycosylase is incorporated into the reaction to prevent PCR product carryover contamination. Finally, RT-PCR-generated amplicons are detected in microtiter wells using virus-specific biotinylated oligoprobes in an enzyme-linked immunosorbent assay-based format. The DNA enzyme immunoassay is based on the capture of PCR product by biotinylated probes fixed onto individual streptavidin-coated wells. Using this method, low levels of NV were detected in stool and both NLV and hepatitis A virus were detected in bivalve mollusks following bioaccumulation. The method also successfully detected NLV in oysters implicated in an outbreak of NLV gastroenteritis. This method dramatically decreases the time needed for analysis and is amenable to automation.

Development of methods to detect "Norwalk-like viruses" (NLVs) and hepatitis A virus in delicatessen foods: application to a food-borne NLV outbreak.

"Norwalk-like viruses" (NLVs) and hepatitis A virus (HAV) are the most common causes of virus-mediated food-borne illness. Epidemiological investigations of outbreaks associated with these viruses have been hindered by the lack of available methods for the detection of NLVs and HAV in foodstuffs. Although reverse transcription (RT)-PCR methods have been useful in detecting NLVs and HAV in bivalve mollusks implicated in outbreaks, to date such methods have not been available for other foods. To address this need, we developed a method to detect NLVs and HAV recovered from food samples. The method involves washing of food samples with a guanidinium-phenol-based reagent, extraction with chloroform, and precipitation in isopropanol. Recovered viral RNA is amplified with HAV- or NLV-specific primers in RT-PCRs, using a viral RNA internal standard control to identify potential sample inhibition. By this method, 10 to 100 PCR units (estimated to be equivalent to 10(2) to 10(3) viral genome copies) of HAV and Norwalk virus seeded onto ham, turkey, and roast beef were detected. The method was applied to food samples implicated in an NLV-associated outbreak at a university cafeteria. Sliced deli ham was positive for a genogroup II NLV as determined by using both polymerase- and capsid-specific primers and probes. Sequence analysis of the PCR-amplified capsid region of the genome indicated that the sequence was identical to the sequence from virus detected in the stools of ill students. The developed method is rapid, simple, and efficient.

Effect of inhibiting HMG-CoA reductase on 7 alpha-hydroxy-4-cholesten-3-one, a marker of bile acid synthesis: contrasting findings in patients with and without prior up-regulation of the latter pathway.

BACKGROUND: Atorvastatin is a potent inhibitor of 3-hydroxy-3-methyl-glutaryl coenzyme A reductase, but its effect on bile acid synthesis is unknown. The objectives of the study were to determine the effect of atorvastatin on bile acid synthesis in patients in whom this process had not been or had been previously up-regulated by pharmacological or surgical means. MATERIALS AND METHODS: Four patients with heterozygous familial hypercholesterolaemia (FH) and partial ileal bypass (PIB) and 19 FH heterozygotes without PIB were treated with placebo, atorvastatin 10 mg and atorvastatin 40 mg daily, each regimen for 4 weeks. The non-PIB group was subsequently treated with bile acid (BA) sequestrant 8-16 g daily followed by co-administration of atorvastatin 10 mg, each for 4 weeks. Plasma 7 alpha-hydroxy-4-cholesten-3-one (7 alpha-HCO), a well-validated marker of BA synthesis was measured using high-performance liquid chromatography with UV detection. RESULTS: The plasma 7 alpha-HCO concentration was tenfold higher with placebo in the PIB than in the non-PIB group (418.5 ng mL-1 vs. 39.6 ng mL-1 p = 0.0001). Levels decreased in PIB patients treated with atorvastatin 10 mg and 40 mg daily (350.1 ng mL-1 and 174.0 ng mL-1, P = 0.007 respectively) but did not change significantly in the non-PIB group (44.7 ng mL-1 and 28.3 ng mL-1 respectively). Administration of BA sequestrant to non-PIB patients increased 7 alpha-HCO to 197.4 ng mL-1; this decreased to 106.0 ng mL-1 during co-administration of atorvastatin 10 mg daily (P = 0.0001). CONCLUSION: Atorvastatin decreases the rate of BA synthesis only if the latter is up-regulated by PIB or BA sequestrants, presumably by limiting the supply of newly synthesized free cholesterol.

Modification of the carbohydrate composition of sulfite pulp by purified and characterized beta-xylanase and beta-xylosidase of Aureobasidium pullulans.

Both beta-xylanase and beta-xylosidase were purified to homogeneity from a xylose-grown culture of Aureobasidium pullulans. Cellular distribution studies of enzyme activities revealed that beta-xylanase was an extracellular enzyme, during both the exponential and stationary phases, whereas beta-xylosidase was mostly periplasmic associated. The beta-xylanase exhibited very high specificity for xylan extracted from Eucalyptus grandis dissolving pulp, whereas the beta-xylosidase was only active on p-nitrophenyl xyloside and xylobiose. Comparison of kcat/Km ratios showed that the beta-xylanase hydrolyzed xylan from dissolving pulp 1.3, 2.1, and 2. 3 times more efficiently than Eucalyptus hemicellulose B, Eucalyptus hemicellulose A, and larchwood xylan, respectively. The beta-xylosidase exhibited a transxylosylation reaction during the hydrolysis of xylobiose. When applied on acid sulfite pulp, both enzymes released xylose and hydrolyzed xylan to a different extent. Although beta-xylosidase (0.4 U/g pulp) liberated more xylose from pulp than beta-xylanase (4.7 U/g pulp), it was responsible for only 3% of xylan solubilization. Treatment of pulp with beta-xylanase liberated 51.7 microgram of xylose/g and hydrolyzed 10% of xylan. The two enzymes acted additively on pulp and removed 12% of pulp xylan. A synergistic effect in terms of release of xylose from pulp was observed when the enzyme mixture of beta-xylanase and beta-xylosidase was supplemented with beta-mannanase. However, this did not result in further enzymatic degradation of pulp xylan. Both beta-xylanase and beta-xylosidase altered the carbohydrate composition of sulfite pulp by increasing the relative cellulose content at the expense of reduced hemicellulose content of pulp.

Distribution of Norwalk virus within shellfish following bioaccumulation and subsequent depuration by detection using RT-PCR.

Consumption of raw bivalve mollusks contaminated with pathogens from human feces continues to present a human health risk. The purpose of this study was to monitor the uptake, localization, and removal of Norwalk virus (NV) in shellfish (oyster and clam) tissues by analyzing virus distribution in selected dissected tissues. Live shellfish were allowed to bioaccumulate different input titers of NV for time periods from 4 to 24 h. In some experiments, depuration by shellfish that bioaccumulated NV and Escherichia coli bacteria was allowed to proceed for 24 or 48 hours. Dissected stomach (St), digestive diverticula (DD), adductor muscle (AM), and hemolymph cells (HC) tissues were assayed for NV by the reverse transcription polymerase chain reaction (RT-PCR) method. An internal RNA standard control was added to the RT-PCR to identify the presence of inhibitors to RT-PCR. NV titers in DD tissues before and after depuration were estimated using quantitative RT-PCR end-point dilution. NV was found in the alimentary tract (DD or St) at all concentrations of input virus, but was present more frequently after exposure to higher levels of virus. NV was detected in AM and HC only following exposure to higher levels of virus. In experiments where depuration by oysters was continued for 48 h, depuration of bacteria was efficient (95% reduction of bacteria), but minimal (7%) reduction of NV titers from DD tissues was detected. These findings indicate that NV can localize both within and outside the alimentary tract of shellfish, and NV is poorly depurated using conditions favorable for E. coli depuration.

Use of heat release and an internal RNA standard control in reverse transcription-PCR detection of Norwalk virus from stool samples.

Norwalk virus (NV) and the Norwalk-like viruses are important human pathogens that cause epidemic acute viral gastroenteritis. Current techniques used to recover NV from clinical samples involve multistep viral extraction and elution procedures with subsequent viral detection by reverse transcription-PCR (RT-PCR). In this study, a simple method using heat to recover viral RNA from 45 stool samples was compared to a conventional viral RNA extraction technique, with subsequent analysis by RT-PCR. In addition, we used an internal RNA standard for the detection of inhibitors present in processed samples. Our results indicate that the use of heat to recover NV RNA from stool samples has a sensitivity for the detection of NV RNA that is similar to the more labor-intensive, time-consuming, conventional RNA extraction technique. The use of an RNA internal standard permits the detection of inhibitors present in processed samples, allowing the identification of false negatives. The standard we developed has the advantage of allowing differential detection between wild-type viral RNA and standard using internal oligoprobe hybridization.

Detection and analysis of a small round-structured virus strain in oysters implicated in an outbreak of acute gastroenteritis.

Outbreaks of shellfish-transmitted viral disease occur periodically, but frequently the causative agent is not identified. In November 1993, during investigation of a multistate outbreak of acute gastroenteritis, incriminated lots of oysters were collected. Oyster tissues (stomachs and digestive diverticula) were processed for virus extraction and nucleic acid purification. Human calicivirus sequences were sought by reverse transcriptase PCR using different primer sets. Amplicons were obtained from 9 of 10 shellfish samples from four different lots when primers specific for the outbreak virus strain were used. The specificity of the amplification was confirmed by hybridization. The amplicons from the nine positive oysters were cloned and sequenced. The sequence of each of the clones was identical to the others but showed some variation (7 of 81 bp) from the sequences obtained from the stools of three persons made III by the outbreak.

Evaluation of a degenerate primer for the PCR detection of human caliciviruses.

Numerous outbreaks of gastroenteritis have been associated with Norwalk virus and Small Round Structured Viruses (SRSVs). These single-stranded RNA viruses, recently classified in the Caliciviridae, have been divided into three genogroups. Antigenic relationships also have been established among the different strains. As both an in vitro culture system and an animal model are lacking for these viruses, virus detection depends primarily on electron microscopy, immunological assays or molecular detection. In this study we first analyzed the genetic homology of the RNA polymerase region for 40 SRSV strains. From a consensus sequence for these strains, we designed a degenerate oligonucleotide to prime cDNA synthesis from viral RNA. We evaluated the degenerate primer in combination with three previously described primers in PCR reactions. A panel of 15 stools containing SRSVs, typed when possible by solid phase immune electron microscopy (SPIEM), were selected to represent all three genogroups and four different SPIEM antigenic types. Serial dilutions of the purified viral nucleic acids were amplified using the three different primer sets. Virus-specific probes were used to characterize the amplicons obtained. Virus-specific amplicons were obtained with at least one primer pair for each strain, but apparent viral RNA titers differed as much as 1000-fold between primer sets. Amplicons from all but one of the 15 strains were confirmed as virus-specific using a panel of 10 different probes. Correlations between the most sensitive primer pair and SPIEM type were seen. This study showed that a single degenerate primer could be used in cDNA synthesis for a variety of SRSVs but that the sensitivity of the RT-PCR assay depended upon the second primer and virus-specific probes used.

Collaborative evaluation of a method for the detection of Norwalk virus in shellfish tissues by PCR.

A multicenter, collaborative trial was performed to evaluate the reliability and reproducibility of a previously described method for the detection of Norwalk virus in shellfish tissues with the PCR (R.L. Atmar, F. H. Neill, J. L. Romalde, F. Le Guyader, C. M. Woodley, T. G. Metcalf, and M. K. Estes, Appl. Environ. Microbiol. 61:3014-3018, 1995). Virus was added to the stomachs and hepatopancreatic tissues of oysters or hard-shell clams in the control laboratory, the samples were shipped to the participating laboratories, and viral nucleic acids were extracted and then detected by reverse transcription-PCR. The sensitivity and specificity of the assay were 85 and 91%, respectively, when results were determined by visual inspection of ethidium bromide-stained agarose gels; the test sensitivity and specificity improved to 87 and 100%, respectively, after confirmation by hybridization with a digoxigenin-labeled, virus-specific probe. We have demonstrated that this method can be implemented successfully by several laboratories to detect Norwalk virus in shellfish tissues.

Rapid and simple assay for feruloyl and p-coumaroyl esterases.

A rapid, simple and sensitive method for detection of ferulic and p-coumaric acids using HPLC has been developed which can be used to determine the respective phenolic acid esterase activities of microorganisms. Prior concentration, purification or derivatization of the samples are not required. As little as 0.5 mg ferulic or p-coumaric acid/I could be detected and estimated in < 1 h. The method is specific for the two phenolic acids sice no interference by other components was observed.

Detection of Norwalk virus and hepatitis A virus in shellfish tissues with the PCR.

A method for the detection of Norwalk virus and hepatitis A virus from shellfish tissues by PCR was developed. Virus was added to the stomach and hepatopancreatic tissues of oysters or hard-shell clams, and viral nucleic acids were purified by a modification of a previously described method (R.L. Atmar, T.G. Metcalf, F.H. Neill, and M.K. Estes, Appl. Environ. Microbiol. 59:631-635, 1993). The new method had the following advantages compared with the previously described method: (i) more rapid sample processing; (ii) increased test sensitivity; (iii) decreased sample-associated interference with reverse transcription-PCR; and (iv) use of chloroform-butanol in place of the chlorofluorocarbon trichlorotrifluoroethane. In addition, internal standards for both Norwalk virus and hepatitis A virus were made which demonstrated when inhibitors to reverse transcription-PCR were present and allowed quantitation of the viral nucleic acids present in samples. This assay can be used to investigate shellfish-associated gastroenteritis outbreaks and to study factors involved in virus persistence in shellfish.

Detection of enteric viruses in oysters by using the polymerase chain reaction.

A procedure for the detection of enteric viral nucleic acid in oysters by the polymerase chain reaction was developed. Known quantities of poliovirus type 1 were seeded into oysters. Virus was extracted and concentrated by using organic flocculation and polyethylene glycol precipitation. Inhibitors of reverse transcription-polymerase chain reaction were present in the oyster extracts, preventing amplification of target viral nucleic acid. The use of cetyltrimethylammonium bromide precipitation sufficiently removed inhibitors to allow detection of as few as 10 PFU of poliovirus. Norwalk virus also could be detected after being seeded into oysters. This methodology may be useful for the detection of these and other shellfish-borne viral pathogens.