A new method of RNA preparation for detection of hepatitis A virus in environmental samples by the polymerase chain reaction

1993 ◽  
Vol 43 (1) ◽  
pp. 77-84 ◽  
Author(s):  
Feng Yang ◽  
Xun Xu
Keyword(s):  
Polymerase Chain Reaction ◽  
Environmental Samples ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
New Method ◽  
Chain Reaction ◽  
Polymerase Chain

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

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.

A Polymerase Chain Reaction–Based Method for the Detection of Hepatitis A Virus in Produce and Shellfish

2002 ◽  
Vol 65 (2) ◽  
pp. 393-402 ◽  
Author(s):  
BISWENDU B. GOSWAMI ◽  
MICHAEL KULKA ◽  
DIANA NGO ◽  
PHILLIP ISTAFANOS ◽  
THOMAS A. CEBULA
Keyword(s):  
Polymerase Chain Reaction ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
General Procedure ◽  
Pcr Analysis ◽  
Genomic Rna ◽  
Chain Reaction ◽  
Viral Origin ◽  
Polymerase Chain ◽  
Test Virus

Outbreaks of gastroenteritis that are suspected to be of viral origin are on the rise. Thus, there is a need for regulatory agencies entrusted with food safety to develop adequate techniques for the detection of viruses in foods. We have established a general procedure for the detection of hepatitis A virus (HAV) in shellfish that, with minor modifications, is also applicable to fresh produce such as cilantro. Total RNA was isolated from shellfish or cilantro, followed by isolation of poly(A)-containing RNA. Because HAV genomic RNA contains a poly(A) tail, the isolation of poly(A)-containing RNA also enriches HAV genomic RNA. Reverse transcription was used to convert the RNA to cDNA, and then amplification was carried out by polymerase chain reaction (PCR). Reamplification with internal primers was used to improve the quality and the quantity of amplified DNA, allowing for post-PCR analysis such as sequence identification of the viral strain. With this procedure, multiple samples could be analyzed in four working days by a single trained individual. The nominal sensitivity of detection of the procedure was 0.15 TCID50 (50% tissue culture infective dose) per 0.62 g of tissue with a test virus. The direct RNA isolation protocol avoided pitfalls associated with whole-virus purification procedures by replacing virus precipitation steps involving polyethylene glycol and Procipitate with phenol extraction. The method is straightforward and reliable. We successfully used this procedure to detect naturally occurring HAV in clams involved in a gastroenteritis outbreak, as well as in cilantro artificially contaminated with a test virus.

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.

Limitations of Molecular Biological Techniques for Assessing the Virological Safety of Foods†

1999 ◽  
Vol 62 (6) ◽  
pp. 691-697 ◽  
Author(s):  
GARY P. RICHARDS
Keyword(s):  
Polymerase Chain Reaction ◽  
Environmental Samples ◽  
Hepatitis A ◽  
Enteric Viruses ◽  
Molecular Techniques ◽  
Technical Expertise ◽  
Chain Reaction ◽  
Assay Sensitivity ◽  
Testing Laboratories ◽  
Polymerase Chain

Enteric viruses, including hepatitis A, Norwalk, and Snow Mountain viruses, Hawaii agent, and rotaviruses have been associated with outbreaks of foodborne illness. Classical culturing procedures are available for poliovirus; however, hepatitis A, Norwalk, and many of the other viruses and agents cannot be propagated in cell culture, therefore, molecular biological tools have emerged as a possible means to detect enteric viruses in foods and environmental samples. There are limitations however in the application of polymerase chain reaction and reverse transcription polymerase chain reaction that restrict their usefulness for measuring the virological safety of foods. The most serious limitation is that molecular techniques fail to discriminate between viable and inactivated viruses even though inactivated viruses pose no threat to the consumer and may be present at levels substantially higher than the virulent forms. Other disadvantages include a lack of assay sensitivity and specificity, high assay costs, and a level of technical expertise not available in most food-testing laboratories. Overall, scientific advances in the development of molecular biological tools have outpaced the demonstration of their validity in assessing the virological safety of foods.

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