Comparison of different primer sets for the RT-PCR detection of hepatitis A virus and astrovirus in mussel tissues

2004 ◽  
Vol 50 (1) ◽  
pp. 131-136 ◽  
Author(s):  
J.L. Romalde ◽  
C. Ribao ◽  
M. Luz Vilariño ◽  
J.L. Barja
Keyword(s):  
Detection Limit ◽  
Critical Point ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Enteric Viruses ◽  
Pcr Detection ◽  
Rt Pcr ◽  
Primer Sets ◽  
Selection Of ◽  
Higher Sensitivity

In the present study, the efficiency of several primer sets for the RT-PCR detection of hepatitis A virus (HAV) and astrovirus from both crude viral extracts and experimentally infected shellfish tissues was evaluated. Differences were observed depending on the primer set employed in the sensitivity of amplification of both viral types. For HAV primers, HAV240/HAV68 yielded the higher sensitivity: showing a detection limit of 0.02-0.1 infectious particles/μL or mg of tissue (either crude extracts or seeded mussel tissues). Regarding detection of AsV, a better performance was observed with primer set A1/A2 achieving a sensitivity of 0.1-1 PFU/μL or mg of tissue. The results obtained in this work strongly indicated that selection of primer sets to be employed for the routine detection of enteric viruses was a critical point in the design of the RT-PCR protocols.

scholarly journals un Identification of enteric viruses from raw water using fluoro-immuno-magnetic separation coupled to RT-PCR

Biomédica ◽  
2021 ◽  
Vol 41 (4) ◽  
Author(s):  
Raquel Villamizar ◽  
Dioselina Peláez-Carvajal ◽  
Luis Felipe Acero
Keyword(s):  
Magnetic Separation ◽  
Water Samples ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Enteric Viruses ◽  
Oral Route ◽  
Human Consumption ◽  
Raw Water ◽  
Rt Pcr ◽  
Contaminated Food

Introduction: Enteric viruses have been associated with the production of a variety of diseases transmitted by the fecal-oral route, carried through contaminated food and water. Given their structure and composition, they are highly resistant to environmental conditions and most of the chemical agents used in the purification processes. Therefore, a systematic monitoring of raw water is necessary to ensure its quality, especially, when it is used as feedstock for the production of drinking water for human consumption. Objective: In the present work the presence of Rotavirus and Hepatitis A Virus was identified by means of the fluoro-immuno-magnetic separation technique (FIMS) in raw water taken from four purification plants in the Norte de Santander department including their water supplies. Materials and methodos: The viruses were captured and separated from the water samples, using magnetic microparticles functionalized with monoclonal anti-Hepatitis A and anti-Rotavirus antibodies. Confocal microscopy was used to monitor the viral concentration process and transmission electron microscopy for morphological visualization of the separated viruses. The reverse transcriptase-coupled polymerase chain reaction (RT-PCR) was applied to confirm the presence of pathogens. Results: The two enteric viruses were identified in most of the analyzed water samples, including their water supply sources. Conclusion: It was possible to determine that the FIMS technique coupled to RT-PCR is highly effective technique in the detection of viral pathogens, in complex matrices such as raw water.

Survival and Persistence of Norovirus, Hepatitis A Virus, and Feline Calicivirus in Marinated Mussels

2004 ◽  
Vol 67 (8) ◽  
pp. 1743-1750 ◽  
Author(s):  
JOANNE HEWITT ◽  
GAIL E. GREENING
Keyword(s):  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Enteric Viruses ◽  
Feline Calicivirus ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Potential Health ◽  
Infectious Dose ◽  
Log Reduction ◽  
Public Health Authorities

Noroviruses (NV) and hepatitis A virus (HAV) are foodborne enteric viruses associated with outbreaks of disease following consumption of raw or lightly cooked bivalve shellfish. Marinated mussels are a popular delicacy, but there is no published information on whether enteric viruses survive the marination process. The survival and persistence of HAV, NV, and a surrogate calicivirus, feline calicivirus (FCV), in marinated mussels over time was determined. NV, HAV, and FCV were inoculated into marinated mussels and marinade liquid and then held at 4°C for up to 4 weeks. Survival of HAV and FCV was quantified by determining the 50% tissue culture infectious dose (TCID50), and these results were correlated with those of the reverse transcription (RT)–PCR assay. The persistence of nonculturable NV was determined by RT–PCR assay only. Over 4 weeks, HAV survived exposure to acid marinade at pH 3.75. There was a 1.7-log reduction in HAV TCID50 titer but no reduction in NV or HAV RT-PCR titer after 4 weeks in marinated mussels. FCV was inactivated in acid conditions although it was still detectable by RT-PCR. To simulate preharvest virus contamination and commercial marination processing, experiments using fresh mussels infected with HAV and NV were performed. HAV and NV persistence was determined using semiquantitative real-time RT-PCR, and HAV infectivity was determined by the TCID50 assay. HAV retained infectivity following simulated commercial marination and exposure to acid conditions over 4 weeks. The survival of pathogenic enteric viruses in marinated mussels constitutes a potential health risk and so is of concern to public health authorities.

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.

scholarly journals Inactivation of Enteric Viruses in Minimally Processed Berries and Herbs

2009 ◽  
Vol 75 (12) ◽  
pp. 4155-4161 ◽  
Author(s):  
S. Butot ◽  
T. Putallaz ◽  
R. Amoroso ◽  
G. Sánchez
Keyword(s):  
Freeze Drying ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Raw Materials ◽  
Enteric Viruses ◽  
Rt Pcr ◽  
Processing Technologies ◽  
Viral Contamination ◽  
Reverse Transcription Pcr ◽  
Dry Heat Treatment

ABSTRACT Several hepatitis A virus (HAV) and human norovirus (HuNoV) outbreaks due to consumption of contaminated berries and vegetables have recently been reported. Model experiments were performed to determine the effectiveness of freeze-drying, freeze-drying combined with heating, and steam blanching for inactivation of enteric viruses that might be present on the surface of berries and herbs. Inactivation of HAV and inactivation of feline calicivirus, a surrogate for HuNoV, were assessed by viral culturing and quantitative reverse transcription PCR (RT-PCR), whereas HuNoV survival was determined only by quantitative RT-PCR. While freeze-drying barely reduced (<1.3 log10 units) the amount of HAV RNA detected in frozen produce, a greater decline in HAV infectivity was observed. The resistance of HuNoV genogroup I (GI) to freeze-drying was significantly higher than that of HuNoV GII on berries. Addition of a terminal dry heat treatment at 120°C after freeze-drying enhanced virus inactivation by at least 2 log10 units, except for HuNoV GII. The results suggest that steam blanching at 95°C for 2.5 min effectively inactivated infectious enteric viruses if they were present in herbs. Our results provide data for adjusting food processing technologies if viral contamination of raw materials is suspected.

Use of a robotic RNA purification protocol based on the NucliSens® easyMAG™ for real-time RT-PCR detection of hepatitis A virus in bottled water

2009 ◽  
Vol 157 (1) ◽  
pp. 80-83 ◽  
Author(s):  
Sylvie Perelle ◽  
Laetitia Cavellini ◽  
Christian Burger ◽  
Sandra Blaise-Boisseau ◽  
Catherine Hennechart-Collette ◽  
...  
Keyword(s):  
Real Time ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Bottled Water ◽  
Pcr Detection ◽  
Rt Pcr ◽  
Rna Purification ◽  
Purification Protocol

RT-PCR evaluation of viral contamination in five shellfish beds over a 21-month period

1998 ◽  
Vol 38 (12) ◽  
pp. 45-50 ◽  
Author(s):  
F. Le Guyader ◽  
L. Miossec ◽  
L. Haugarreau ◽  
E. Dubois ◽  
H. Kopecka ◽  
...  
Keyword(s):  
Hepatitis A ◽  
Microbial Contamination ◽  
Hepatitis A Virus ◽  
Enteric Viruses ◽  
Faecal Coliform ◽  
Faecal Coliforms ◽  
Rt Pcr ◽  
Viral Contamination

Five shellfish beds were sampled for 21 months and evaluated for microbial contamination. Viral extraction was performed on dissected tissues and the clinically most important enteric viruses (hepatitis A virus, small round structured virus, rotavirus and enterovirus) were searched for by RT-PCR and hybridization. Among the 104 samples analysed, 66% were contaminated by at least one virus and 34% were negative for any virus. The two sites regularly contaminated by faecal coliforms had the highest percentage of viral contamination and HAV was detected only in these sites. However, sampling sites meeting the criteria for commercialisation showed occasional viral contamination and viruses were detected in samples with no faecal coliform contamination.

Sign in / Sign up

Export Citation Format

Share Document