scholarly journals A Multiplex Reverse Transcription-PCR Method for Detection of Human Enteric Viruses in Groundwater

2003 ◽  
Vol 69 (6) ◽  
pp. 3158-3164 ◽  
Author(s):  
G. Shay Fout ◽  
Beth C. Martinson ◽  
Michael W. N. Moyer ◽  
Daniel R. Dahling
Keyword(s):  
Reverse Transcription ◽  
Hepatitis A Virus ◽  
Disease Outbreaks ◽  
Enteric Viruses ◽  
The United States ◽  
Norwalk Virus ◽  
Rt Pcr ◽  
Reverse Transcription Pcr ◽  
Groundwater Samples ◽  
Human Enteric Viruses

ABSTRACT Untreated groundwater is responsible for about half of the waterborne disease outbreaks in the United States. Human enteric viruses are thought to be leading etiological agents of many of these outbreaks, but there is relatively little information on the types and levels of viruses found in groundwater. To address this problem, monthly samples from 29 groundwater sites were analyzed for 1 year for enteroviruses, hepatitis A virus, Norwalk virus, reoviruses, and rotaviruses by multiplex reverse transcription-PCR (RT-PCR). A procedure with which to remove environmental RT-PCR inhibitors from groundwater samples was developed. The procedure allowed an average of 71 liters of the original groundwater to be assayed per RT-PCR, with an average virus recovery rate of 74%, based on seeded samples. Human enteric viruses were detected in 16% of the groundwater samples analyzed, with reoviruses being the most frequently detected virus group.

scholarly journals Three-Year Study To Assess Human Enteric Viruses in Shellfish

2000 ◽  
Vol 66 (8) ◽  
pp. 3241-3248 ◽  
Author(s):  
F. Le Guyader ◽  
L. Haugarreau ◽  
L. Miossec ◽  
E. Dubois ◽  
M. Pommepuy
Keyword(s):  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Virus Detection ◽  
Enteric Viruses ◽  
Molecular Technique ◽  
Norwalk Virus ◽  
Viral Contamination ◽  
Long Period ◽  
Reverse Transcription Pcr ◽  
Human Enteric Viruses

ABSTRACT The main pathogenic enteric viruses able to persist in the environment, such as hepatitis A virus (HAV), Norwalk-like virus (NLV), enterovirus (EV), rotavirus (RV), and astrovirus (AV), were detected by reverse transcription-PCR and hybridization in shellfish during a 3-year study. Oyster samples (n = 108), occasionally containing bacteria, were less frequently contaminated, showing positivity for AV (17%), NLV (23%), EV (19%), and RV (27%), whereas mussel samples, collected in areas routinely impacted by human sewage, were more highly contaminated: AV (50%), HAV (13%), NLV (35%), EV (45%), and RV (52%). Sequences obtained from HAV and NLV amplicons showed a great variety of strains, especially for NLV (strains close to Mexico, Snow Mountain Agent, or Norwalk virus). Viral contamination was mainly observed during winter months, although there were some seasonal differences among the viruses. This first study of virus detection over a fairly long period of time suggests that routine analysis of shellfish by a molecular technique is feasible.

scholarly journals Incidence of Enteric Viruses in Groundwater from Household Wells in Wisconsin

2003 ◽  
Vol 69 (2) ◽  
pp. 1172-1180 ◽  
Author(s):  
Mark A. Borchardt ◽  
Phil D. Bertz ◽  
Susan K. Spencer ◽  
David A. Battigelli
Keyword(s):  
Hepatitis A Virus ◽  
Enteric Viruses ◽  
The United States ◽  
Land Application ◽  
Private Household ◽  
Septic Systems ◽  
Rt Pcr ◽  
Sequential Samples ◽  
Human Enteric Viruses ◽  
Public Water Systems

ABSTRACT Recent studies on the contamination of groundwater with human enteric viruses have focused on public water systems, whereas little is known about the occurrence of viruses in private household wells. The objective of the present study was to estimate the incidence of viruses in Wisconsin household wells located near septage land application sites or in rural subdivisions served by septic systems. Fifty wells in seven hydrogeologic districts were sampled four times over a year, once each season. Reverse transcriptase PCR (RT-PCR), followed by Southern hybridization, was used to detect enteroviruses, rotavirus, hepatitis A virus (HAV), and Norwalk-like viruses (NLVs). In addition, cell culture was used to detect culturable enteroviruses. Companion water samples were collected for total coliforms, Escherichia coli, fecal enterococci, F-specific RNA coliphages, nitrate, and chloride analyses. Among the 50 wells, four (8%) were positive for viruses by RT-PCR. Three wells were positive for HAV, and the fourth well was positive for both rotavirus and NLV in one sample and an enterovirus in another sample. Contamination was transient, since none of the wells was virus positive for two sequential samples. Culturable enteroviruses were not detected in any of the wells. Water quality indicators were not statistically associated with virus occurrence, although some concordance was noted for chloride. The present study is the first in the United States to systematically monitor private household wells for virus contamination and, combined with data for public wells, provides further insight on the extent of groundwater contamination with human enteric viruses.

Detection Methods for Human Enteric Viruses in Representative Foods†

2000 ◽  
Vol 63 (12) ◽  
pp. 1738-1744 ◽  
Author(s):  
PARIS R. LEGGITT ◽  
LEE-ANN JAYKUS
Keyword(s):  
Hepatitis A Virus ◽  
Enteric Viruses ◽  
Food Sample ◽  
Viral Rna ◽  
Detection Methods ◽  
Norwalk Virus ◽  
Rt Pcr ◽  
Viral Contamination ◽  
Initial Inoculum ◽  
Human Enteric Viruses

Although viral foodborne disease is a significant problem, foods are rarely tested for viral contamination, and when done, testing is limited to shellfish commodities. In this work, we report a method to extract and detect human enteric viruses from alternative food commodities using an elution-concentration approach followed by detection using reverse transcription-polymerase chain reaction (RT-PCR). Fifty-gram lettuce or hamburger samples were artificially inoculated with poliovirus type 1 (PV1), hepatitis A virus (HAV), or the Norwalk virus and processed by the sequential steps of homogenization, filtration, Freon extraction (hamburger), and polyethylene glycol (PEG) precipitation. To reduce volumes further and remove RT-PCR inhibitors, a secondary PEG precipitation was necessary, resulting in an overall 10- to 20-fold sample size reduction from 50 g to 3 to 5 ml. Virus recoveries in secondary PEG concentrates ranged from 10 to 70% for PV1 and 2 to 4% for HAV as evaluated by mammalian cell culture infectivity assay. Total RNA from PEG concentrates was extracted to a small volume (30 to 40 μl) and subjected to RT-PCR amplification of viral RNA sequences. Detection limit studies indicated that viral RNA was consistently detected by RT-PCR at initial inoculum levels ≥102 PFU/50-g food sample for PV1 and ≥103 PFU/50-g food sample for HAV. In similar studies with the Norwalk virus, detection at inoculum levels ≥1.5 × 103 PCR-amplifiable units/50-g sample for both food products was possible. All RT-PCR amplicons were confirmed by subsequent Southern hybridization. The procedure reported represents progress toward the development of methods to detect human enteric viral contamination in foods other than shellfish.

Virion Concentration Method for the Detection of Human Enteric Viruses in Extracts of Hard-Shelled Clams†

1998 ◽  
Vol 61 (4) ◽  
pp. 458-465 ◽  
Author(s):  
ALISSA B. DIX ◽  
LEE-ANN JAYKUS
Keyword(s):  
Cell Culture ◽  
Hepatitis A Virus ◽  
Direct Detection ◽  
Enteric Viruses ◽  
Precipitation Method ◽  
Norwalk Virus ◽  
Rt Pcr ◽  
Genomic Amplification ◽  
Initial Inoculum ◽  
Human Enteric Viruses

A method to extract and concentrate intact human enteric viruses from oyster extracts for detection using reverse transcription-polymerase chain reaction (RT-PCR) was applied to hard-shelled clams (Mercenaria mercenaria). Fifty-gram clam samples were processed by an adsorption-elution-precipitation method and then seeded with 101 to 105 PFU of poliovirus 1 (PV1) and/or hepatitis A virus (HAV). Seeded viruses in extracts were purified by fluorocarbon (Freon) extraction and concentrated by polyethylene glycol (PEG) precipitation and elution. Efficiency of virion recovery from PEG precipitates was dependent upon PEG concentration and elution buffer volume, with optimized variables yielding recoveries as high as 99% for PV1 and 45% for FIAV, as evaluated by cell culture infectivity assay. To further concentrate viruses, remove inhibitors, and reduce sample volumes, the protein-precipitating agent Pro-Cipitate was used in an adsorption-elution-precipitation scheme. The final concentrate was of low volume (<1 ml) and directly compatible with viral genomic amplification using RT-PCR. When extracts from 50-g clam samples were seeded and processed by the combined concentration and purification scheme, direct RT-PCR detection of viral genomic RNA was possible at initial inoculum levels of 103 PFU for PV1 and HAV. Corresponding virus recoveries based on cell culture infectivity were 7 to 50% and 0.3 to 8% for PV1 and HAV, respectively. When extracts of clams were artificially contaminated with the Norwalk virus, direct detection of virion RNA using RT-PCR and subsequent oligoprobe hybridization was possible at levels as low as 450 RT-PCR amplifiable units of the Norwalk virus per extract of 50-g clam sample.

scholarly journals Quantification of Hepatitis A Virus in Shellfish by Competitive Reverse Transcription-PCR with Coextraction of Standard RNA

1999 ◽  
Vol 65 (1) ◽  
pp. 322-326 ◽  
Author(s):  
Charlotte Arnal ◽  
Virginie Ferre-Aubineau ◽  
Berangere Mignotte ◽  
Berthe Marie Imbert-Marcille ◽  
Sylviane Billaudel
Keyword(s):  
Tissue Culture ◽  
Reverse Transcription ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Internal Standard ◽  
Wild Type ◽  
Rt Pcr ◽  
Reverse Transcription Pcr ◽  
Reproducible Method ◽  
Dna Enzyme Immunoassay

ABSTRACT To quantify hepatitis A virus (HAV) in experimentally contaminated mussels, we developed an internal standard RNA with a 7-nucleotide deletion for competitive reverse transcription (RT)-PCR. Deposited directly into the sample, this standard was used both as extraction control and as quantification tool. After coextraction and competitive RT-PCR, standard and wild-type products were detected by differential hybridization with specific probes and a DNA enzyme immunoassay. The quantifiable range with this reproducible method was 104 to 107 copies of HAV/gram or 400 to 106 50% tissue culture infective doses/ml.

A Multiplex Reverse Transcription Polymerase Chain Reaction Method for the Detection of Foodborne Viruses†

1999 ◽  
Vol 62 (10) ◽  
pp. 1210-1214 ◽  
Author(s):  
SORAYA I. ROSENFIELD ◽  
LEE-ANN JAYKUS
Keyword(s):  
Polymerase Chain Reaction ◽  
Reverse Transcription ◽  
Hepatitis A Virus ◽  
Simultaneous Detection ◽  
Polymerase Chain Reaction Method ◽  
Human Enterovirus ◽  
Norwalk Virus ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Polymerase Chain

A multiplex reverse transcription polymerase chain reaction (RT-PCR) method was developed for the simultaneous detection of the human enteroviruses, hepatitis A virus (HAV) and Norwalk virus (NV). Poliovirus type 1 (PV1) was chosen as a model for the human enterovirus group. Three different sets of primers were used to produce three size-specific amplicons of 435 bp, 270 bp, and 192 bp for PV1, NV, and HAV, respectively. RT-PCR products were separated by agarose gel electrophoresis, and amplicon identity was confirmed by Southern transfer followed by DNA hybridization using nonradio-active, digoxigenin-labeled internal probes. When tested on mixed, purified virus suspensions, the multiplex method achieved detection limits of ≤1 infectious unit (PV1 and HAV) or RT-PCR-amplifiable unit (NV) for all viruses. With further streamlining efforts such as single tube amplification and liquid hybridization, multiplex PCR offers advantages over cell culture methodology and monoplex PCR because it allows for rapid and cost-effective detection of several human enteric viruses in a single reaction tube.

Detection of Human Enteric Viruses in Japanese Clams

2008 ◽  
Vol 71 (8) ◽  
pp. 1689-1695 ◽  
Author(s):  
GRANT S. HANSMAN ◽  
TOMOICHIRO OKA ◽  
TIAN-CHENG LI ◽  
OSAMU NISHIO ◽  
MAMORU NODA ◽  
...  
Keyword(s):  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Enteric Viruses ◽  
Aichi Virus ◽  
Human Consumption ◽  
Reverse Transcription Pcr ◽  
Different Types ◽  
Group A ◽  
Human Enteric Viruses ◽  
High Level

A total of 57 clam packages that were collected from supermarkets and fish markets from 11 different sites in western Japan between 8 December 2005 and 6 September 2006 were examined for human enteric viruses (i.e., norovirus, Aichi virus, rotavirus, adenovirus, hepatitis A virus, and astrovirus), using PCR and reverse transcription PCR. Sixty-one percent of the packages were contaminated with one type of virus, 9% had two different types of viruses, 28% had three different types of viruses, and 9% had at least four different types of viruses. Thirty-one (54%) of 57 packages were contaminated with noroviruses. Norovirus genogroup I and genogroup II sequences were detected in 24 and 23 packages, respectively, and these sequences belonged to nine genogroup I and eight genogroup II genotypes. Aichi viruses were found in 19 (33%) of 57 packages, and these belonged to genogroup A. Rotaviruses (group A) were detected in 14 (42%) of 33 of packages and 9 of 14 rotavirus-positive packages contained two or more rotavirus genogroup types. Adenoviruses (Ad40 and Ad41) were detected in 17 (52%) of 33 packages. One of the 57 (2%) packages was positive with hepatitis A virus (subtype IA). Astrovirus was not detected in any of the packages. This is the first study to detect such a high level of contamination in Japanese clams. These results represent an important finding because the Japanese clams were considered suitable for human consumption. Further studies are needed to determine the health risks associated with eating these highly contaminated clams.

Viral Foodborne Disease Agents of Concern

1994 ◽  
Vol 57 (2) ◽  
pp. 176-178 ◽  
Author(s):  
DEAN O. CLIVER
Keyword(s):  
United States ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
The United States ◽  
Molecular Techniques ◽  
Detection Methods ◽  
Foodborne Disease ◽  
Norwalk Virus ◽  
Routine Basis ◽  
Human Enteric Viruses

Viruses transmitted to humans via foods generally emanate from the human intestines. In the United States, Norwalk virus ranked #5, hepatitis A virus #6, and “other viruses” (principally rotavirus) #10 among the top 10 causes of foodborne disease during 1983–1987. Molluscs are the most frequently reported vehicles, but any food handled by humans may transmit human enteric viruses. Some fruit and vegetable vehicles may have been contaminated in the field before or during harvesting. Viruses in foods may be inactivated before the food is eaten, and thus, not cause infection. Increasingly sensitive detection methods, largely based on “molecular” techniques, are becoming available for these viruses but are not applicable to monitoring foods on a routine basis.

scholarly journals Procedure for Rapid Concentration and Detection of Enteric Viruses from Berries and Vegetables

2006 ◽  
Vol 73 (1) ◽  
pp. 186-192 ◽  
Author(s):  
S. Butot ◽  
T. Putallaz ◽  
G. Sánchez
Keyword(s):  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Detection Limits ◽  
Enteric Viruses ◽  
Rt Pcr ◽  
Specific Primers ◽  
Reverse Transcription Pcr ◽  
Concentration Method ◽  
Elution Buffer ◽  
Elution Method

ABSTRACT Several hepatitis A virus (HAV) and norovirus (NV) outbreaks due to consumption of berries and vegetables have been reported during recent years. To facilitate the detection of enteric viruses that may be present on different fresh and frozen products, we developed a rapid and sensitive detection method for HAV, NV, and rotavirus (RV). Initial experiments focused on optimizing the composition of the elution buffer, improving the viral concentration method, and evaluating the performance of various extraction kits. Viruses were extracted from the food surface by a direct elution method in a glycine-Tris (pH 9.5) buffer containing 1% beef extract and concentrated by ultrafiltration. Occasionally, PCR inhibitors were present in the processed berry samples, which gave relatively poor detection limits. However, this problem was overcome by adding a pectinase treatment in the protocol, which markedly improved the sensitivity of the method. After optimization, this concentration method was applied in combination with real-time reverse transcription-PCR (RT-PCR) using specific primers in various types of berries and vegetables. The average detection limits were 1 50% tissue culture infective dose (TCID50), 54 RT-PCR units, and 0.02 TCID50 per 15 g of food for HAV, NV, and RV, respectively. Based on our results, it is concluded that this procedure is suitable to detect and quantify enteric viruses within 6 h and can be applied for surveillance of enteric viruses in fresh and frozen products.

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