Development of PCR Methods for Enteric Virus Detection in Water

1993 ◽  
Vol 27 (3-4) ◽  
pp. 211-218 ◽  
Author(s):  
K. J. Schwab ◽  
R. De Leon ◽  
M. D. Sobsey
Keyword(s):  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Detection System ◽  
Virus Detection ◽  
Enteric Virus ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Sample Concentration ◽  
Spin Column ◽  
Polymerase Chain

This study developed a reverse transcriptase-polymerase chain reaction (RT-PCR) detection system for enteric viruses in sample concentrates obtained by conventional filter adsorption-elution methods. One liter beef extract (BE)-glycine (G) eluant seeded with poliovirus 1 and hepatitis A virus (HAV) was used as a model system and the eluant further processed for RT-PCR compatibility. Sample concentration and purification procedures consisted of polyethylene glycol (PEG) precipitation, Pro-Cipitate (Affinity Technology, New Brunswick, NJ) precipitation, a second PEG precipitation, spin-column chromatography, and ultrafiltration. Sample volumes are reduced from 1 L to 20-40 µL and purified sufficiently for viral detection by RT-PCR. As little as 3 PFU of poliovirus 1 in an initial 1 L eluate were detected by RT-PCR.

Hepatitis A Virus Detection in Oysters (Crassostrea gigas) in Santa Catarina State, Brazil, by Reverse Transcription–Polymerase Chain Reaction

2003 ◽  
Vol 66 (3) ◽  
pp. 507-511 ◽  
Author(s):  
C. COELHO ◽  
A. P. HEINERT ◽  
C. M. O. SIMÕES ◽  
C. R. M. BARARDI
Keyword(s):  
Polymerase Chain Reaction ◽  
Reverse Transcription ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Severe Illness ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Santa Catarina ◽  
Polymerase Chain ◽  
Santa Catarina State

Shellfish are readily contaminated with viruses present in water containing sewage because of the concentration effect of filter feeding. Hepatitis A virus (HAV) is the main cause of acute hepatitis worldwide and may lead to severe illness or even death. It is transmitted through fecal and oral routes and causes widespread endemic and asymptomatic infections in young children. Here we describe a method for the detection of HAV RNA in shellfish involving the extraction of total RNA from oyster meat followed by reverse transcription–polymerase chain reaction (RT-PCR). Virus recovery from oyster extracts artificially seeded with HAV strain HM 175 was examined by RT-PCR. The minimum detection limit was 3.3 focus-forming units of HAV, and the recovery rate was 75.7%. This method was used to assess the viral contamination of four shellfish beds in Santa Catarina State, Brazil, over a 1-year period. Six (22%) of 27 samples collected in autumn and winter from one shellfish bed tested positive for HAV.

ICC/PCR detection of enteroviruses and hepatitis A virus in environmental samples

2001 ◽  
Vol 47 (2) ◽  
pp. 153-157 ◽  
Author(s):  
Kelly A Reynolds ◽  
Charles P Gerba ◽  
Morteza Abbaszadegan ◽  
Ian L Pepper
Keyword(s):  
Cell Culture ◽  
Environmental Samples ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Virus Detection ◽  
Rt Pcr ◽  
Specific Detection ◽  
Polymerase Chain ◽  
Conventional Cell ◽  
Polymerase Chain Reaction Methodology

This study applied the integrated cell culture/polymerase chain reaction methodology (ICC/PCR) for rapid and specific detection of both cytopathogenic and noncytopathogenic viruses. Results of this study showed that the use of direct RT-PCR or conventional cell culture alone may yield erroneous results with the analysis of environmental samples. The purpose of this study was to compare cultural, molecular, and combined assays for the most effective method of virus detection in variable environmental samples. Using ICC/PCR, stock enterovirus inocula of [Formula: see text]10 PFU were PCR positive in at least 4/5 replicate flasks after only 5 h of incubation in cell culture, and in all flasks after [Formula: see text]10 h. An inoculum of one PFU was detected by PCR after 20 h of cell culture incubation while for concentrations of virus below one PFU, 25 h of incubation was sufficient. Similarly, hepatitis A virus (HAV) inocula of 100 MPN/flask, produced indeterminate CPE in cell culture, but were clearly detected by ICC/PCR following 48 h of incubation. Lower levels of HAV, 1 and 10 MPN, were detected by ICC/PCR after 96 to 72 h of incubation, respectively. Cell culture lysates from 11 environmental sample concentrates of sewage, marine water, and surface drinking water sources, were positive for enteroviruses by ICC/PCR compared to 3 positive by direct RT-PCR alone. Results from ICC/PCR eventually agreed with cell culture but required [Formula: see text]48 h of incubation, compared to as long as 3 weeks for CPE following incubation with BGM and FRhK cells.Key words: RT-PCR, cell culture, ICC/PCR, enterovirus, hepatitis A virus.

scholarly journals An outbreak of hepatitis A among homosexuals linked to a family outbreak

2002 ◽  
Vol 129 (1) ◽  
pp. 113-117 ◽  
Author(s):  
K. STENE-JOHANSEN ◽  
P. A. JENUM ◽  
T. HOEL ◽  
H. BLYSTAD ◽  
H. SUNDE ◽  
...  
Keyword(s):  
Molecular Epidemiology ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Sequence Information ◽  
Risk Of Infection ◽  
Rt Pcr ◽  
Chain Reaction ◽  
The Family ◽  
Polymerase Chain ◽  
Family Outbreak

Several outbreaks of hepatitis A occurred in Norway in 1995–8. Molecular epidemiology was used to follow the spread of hepatitis A virus in the population. Distinct strains of hepatitis A virus (HAV) were detected by reverse transcriptase–polymerase chain reaction (RT–PCR) and subsequent sequencing in serum from patients in different communities at risk of infection. Two HAV strains were detected in an outbreak among 26 men having sexual contact with other men. One of these strains was also detected in a geographically limited family outbreak. The family outbreak was first believed to be acquired abroad. The sequence information linked the two outbreaks, and epidemiological and serological analyses revealed the transmission route. This study demonstrates the importance of molecular epidemiology in outbreak investigation, surveillance and monitoring of hepatitis A in the population.

scholarly journals Prolonged fecal excretion of hepatitis A virus in adult patients with hepatitis A as determined by polymerase chain reaction

Hepatology ◽  
1996 ◽  
Vol 24 (1) ◽  
pp. 10-13 ◽  
Author(s):  
H Yotsuyanagi ◽  
K Koike ◽  
K Yasuda ◽  
K Moriya ◽  
Y Shintani ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Adult Patients ◽  
Fecal Excretion ◽  
Chain Reaction ◽  
Polymerase Chain

scholarly journals Detection of multiple enteric virus strains within a foodborne outbreak of gastroenteritis: an indication of the source of contamination

2004 ◽  
Vol 133 (1) ◽  
pp. 41-47 ◽  
Author(s):  
C. I. GALLIMORE ◽  
C. PIPKIN ◽  
H. SHRIMPTON ◽  
A. D. GREEN ◽  
Y. PICKFORD ◽  
...  
Keyword(s):  
Arabian Gulf ◽  
Enteric Virus ◽  
Multiple Infections ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Epidemiological Analysis ◽  
Viral Aetiology ◽  
Polymerase Chain ◽  
Most Probable Explanation ◽  
Virus Strains

An outbreak of acute gastroenteritis of suspected viral aetiology occurred in April 2003 in the British Royal Fleet Auxillary ship (RFA) Argus deployed in the Northern Arabian Gulf. There were 37 cases amongst a crew of 400 personnel. Of 13 samples examined from cases amongst the crew, six enteric viruses were detected by reverse transcriptase polymerase chain reaction (RT–PCR). Five different viruses were identified including, three norovirus genotypes, a sapovirus and a rotavirus. No multiple infections were detected. A common food source was implicated in the outbreak and epidemiological analysis showed a statistically significant association with salad as the source of the outbreak, with a relative risk of 3·41 (95% confidence interval of 1·7–6·81) of eating salad on a particular date prior to the onset of symptoms. Faecal contamination of the salad at source was the most probable explanation for the diversity of viruses detected and characterized.

Persistence of Hepatitis A Virus in Oysters†

2003 ◽  
Vol 66 (2) ◽  
pp. 331-334 ◽  
Author(s):  
DAVID H. KINGSLEY ◽  
GARY P. RICHARDS
Keyword(s):  
Polymerase Chain Reaction ◽  
Crassostrea Virginica ◽  
Reverse Transcription ◽  
Hepatitis A ◽  
Infectious Virus ◽  
Hepatitis A Virus ◽  
Complete Removal ◽  
Chain Reaction ◽  
Daily Feeding ◽  
Polymerase Chain

We investigated the ability of hepatitis A virus (HAV) to persist for up to 6 weeks in Eastern oysters (Crassostrea virginica). Viral RNA was detected by reverse transcription–polymerase chain reaction 6 weeks after 16 h of exposure to 90,000 PFU (180 PFU/ml of seawater) of HAV. Assaying for infectious virus in oysters that received a daily feeding of phytoplankton recovered 3,800, 650, and 500 PFU of HAV 1, 2, and 3 weeks after contamination with 90,000 PFU of HAV, respectively. However, no infectious HAV was isolated from oysters 4, 5, or 6 weeks after contamination. These results support the position that shellfish depuration is insufficient for the complete removal of infectious viruses. Extended relay times (in excess of 4 weeks) may be required to produce virologically safe shellfish.

scholarly journals Improved RNA Extraction from Woody Plants for the Detection of Viral Pathogens by Reverse Transcription-Polymerase Chain Reaction

Plant Disease ◽  
1997 ◽  
Vol 81 (2) ◽  
pp. 222-226 ◽  
Author(s):  
Donald J. MacKenzie ◽  
Morven A. McLean ◽  
Srima Mukerji ◽  
Margaret Green
Keyword(s):  
Polymerase Chain Reaction ◽  
Reverse Transcription ◽  
Woody Plants ◽  
Rna Extraction ◽  
Viral Rna ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Apple Stem ◽  
Spin Column ◽  
Polymerase Chain

An efficient procedure for the extraction of high-quality RNA from woody plants without the use of phenol, organic solvents, or alcohol precipitation is described. The method employs commercially available spin-column matrices and mitigates the inhibitory effects of plant polysaccharides and polyphenolic compounds commonly observed on subsequent polymerase chain reaction amplification when conventional extraction methods are applied to woody plant species. The method described has been successfully used in the development of highly sensitive reverse transcription-polymerase chain reaction (RT-PCR) techniques for the detection of a number of viruses in their woody hosts. The viruses detected included apple stem grooving capillovirus (ASGV), apple stem pitting virus, Prunus necrotic ringspot ilarvirus (PNRSV), grapevine fanleaf and Arabis mosaic nepoviruses, and grapevine leafroll-associated closterovirus type 3. The method described was equally effective for the extraction of viral RNA from either budwood, leaves, or flower blossoms as determined by the equivalent RT-PCR detection of ASGV and PNRSV from these tissues. Detection of viral RNA in samples of total plant RNA prepared using this method was found to be as sensitive as was previously described for the immunocapture RT-PCR technique.

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