scholarly journals Mechanisms of Inactivation of Hepatitis A Virus by Chlorine

2002 ◽  
Vol 68 (10) ◽  
pp. 4951-4955 ◽  
Author(s):  
Jun Wen Li ◽  
Zhong Tao Xin ◽  
Xin Wei Wang ◽  
Jin Lai Zheng ◽  
Fu Huan Chao
Keyword(s):  
Cell Culture ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Specific Region ◽  
Enzyme Linked Immunosorbent Assay ◽  
Coding Region ◽  
Rt Pcr ◽  
Entire Genome ◽  
Reverse Transcription Pcr ◽  
Complete Inactivation

ABSTRACT The study was intended to investigate the feasibility of reverse transcription-PCR (RT-PCR) for evaluation of the efficacy of inactivation of viruses in water and to elucidate the mechanisms of inactivation of hepatitis A virus (HAV) by chlorine. Cell culture, enzyme-linked immunosorbent assay, and long-overlap RT-PCR were used to detect the infectivity, antigenicity, and entire genome of HAV inactivated or destroyed by chlorine. The cell culture results revealed the complete inactivation of infectivity after 30 min of exposure to 10 or 20 mg of chlorine per liter and the highest level of sensitivity in the 5′ nontranslated regions (5′NTR), inactivation of which took as much time as the inactivation of infectivity of HAV by chlorine. However, antigenicity was not completely destroyed under these conditions. Some fractions in the coding region were resistant to chlorine. To determine the specific region of the 5′NTR lost, three segments of primers were redesigned to monitor the region from bp 1 to 1023 across the entire genome. It was shown that the sequence from bp 1 to 671 was the region most sensitive to chlorine. The results suggested that the inactivation of HAV by chlorine was due to the loss of the 5′NTR. It is believed that PCR can be used to assess the efficacy of disinfection of HAV by chlorine as well as to research the mechanisms of inactivation of viruses by disinfectants.

Rapid Detection Method for Hepatitis A Virus from Lettuce by a Combination of Filtration and Integrated Cell Culture–Real-Time Reverse Transcription PCR

2011 ◽  
Vol 74 (10) ◽  
pp. 1756-1761 ◽  
Author(s):  
JI-YEON HYEON ◽  
JUNG-WHAN CHON ◽  
CHANKYU PARK ◽  
JUNG-BOK LEE ◽  
IN-SOO CHOI ◽  
...  
Keyword(s):  
Cell Culture ◽  
Real Time ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Rt Pcr ◽  
Cytopathic Effects ◽  
Reverse Transcription Pcr ◽  
Charged Membrane ◽  
Polyethylene Glycol Precipitation ◽  
Positively Charged

We have developed a rapid and simple method for filtration using a positively charged membrane to concentrate hepatitis A virus (HAV) from lettuce and an integrated cell culture–real-time reverse transcription PCR (ICC–real-time RT-PCR) to detect infectious HAV. The most suitable buffer for HAV concentration by filtration was 100 mM Tris-HCl, 50 mM glycine (pH 9.5). Filtration using the NanoCeram matrix was compared with polyethylene glycol precipitation for viral concentration from lettuce inoculated with 6 log RNA copies of HAV. The recovery rate of filtration was statistically higher than that of polyethylene glycol precipitation (47.3 versus 24.9%, respectively). The sensitivity of ICC–real-time RT-PCR for detection of infectious HAV was determined by inoculation of FRhK-4 cells with HAV (4 log to 0 log RNA copies). ICC–real-time RT-PCR detected infectious HAV on average 5 days earlier than cytopathic effects at all inoculation levels. HAV recovered from lettuce (approximately 3 log RNA copies) was also analyzed with ICC–real-time RT-PCR. Infectious HAV was detected within 2 days postinfection by ICC–real-time RT-PCR, whereas cytopathic effects were not observed until 7 days postinfection. Coupled with a virus concentration and purification system using a positively charged membrane, ICC–real-time RT-PCR has the potential to become a novel and rapid method for the detection of infectious HAV in vegetables.

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.

Evaluation of a Sensitive Reverse Transcription PCR–Enzyme-Linked Immunosorbent Assay for Detection of Hepatitis A Virus in Oysters (Saccostrea glomerata) on the East Coast of the Gulf of Thailand

2014 ◽  
Vol 77 (5) ◽  
pp. 859-863 ◽  
Author(s):  
URAIWAN INTAMASO ◽  
SITTHISAK KETKHUNTHOD
Keyword(s):  
Immunosorbent Assay ◽  
Reverse Transcription ◽  
Hepatitis A ◽  
East Coast ◽  
Hepatitis A Virus ◽  
Detection Sensitivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Gulf Of Thailand ◽  
Rt Pcr ◽  
The Gulf Of Thailand

Hepatitis A virus (HAV) contamination in food can lead to major health problems. We developed a combination reverse transcription (RT) PCR method plus enzyme-linked immunosorbent assay (ELISA) to detect HAV in fresh oysters harvested along the east coast of the Gulf of Thailand. Viral nucleic acid was extracted via the glycine–arginine–polyethylene glycol method followed by RT-PCR amplification with specifically designed primers against HAV and an ELISA to detect the digoxigenin-labeled RT-PCR products. The ELISA in concert with the RT-PCR protocol further increased the detection sensitivity by 100-fold for the HAV genome and 10-fold in artificially contaminated oysters. The overall sensitivity of the RT-PCR in combination with the ELISA was 31.88 pg and 16 PFU/g, respectively. The ELISA increases the specificity of the RT-PCR assay for detecting naturally occurring HAV in oysters. This combined RT-PCR-ELISA approach is a practical and sensitive method for HAV detection and can be utilized in routine screening for HAV in shellfish.

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.

scholarly journals Detection of Hepatitis A Virus by the Nucleic Acid Sequence-Based Amplification Technique and Comparison with Reverse Transcription-PCR

2001 ◽  
Vol 67 (12) ◽  
pp. 5593-5600 ◽  
Author(s):  
Julie Jean ◽  
Burton Blais ◽  
André Darveau ◽  
Ismaı̈l Fliss
Keyword(s):  
Nucleic Acid ◽  
Reverse Transcription ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Nucleic Acid Sequence ◽  
Clinical Samples ◽  
Rt Pcr ◽  
Dot Blot ◽  
Reverse Transcription Pcr ◽  
Dot Blot Assay

ABSTRACT A nucleic acid sequence-based amplification (NASBA) technique for the detection of hepatitis A virus (HAV) in foods was developed and compared to the traditional reverse transcription (RT)-PCR technique. Oligonucleotide primers targeting the VP1 and VP2 genes encoding the major HAV capsid proteins were used for the amplification of viral RNA in an isothermal process resulting in the accumulation of RNA amplicons. Amplicons were detected by hybridization with a digoxigenin-labeled oligonucleotide probe in a dot blot assay format. Using the NASBA, as little as 0.4 ng of target RNA/ml was detected per comparison to 4 ng/ml for RT-PCR. When crude HAV viral lysate was used, a detection limit of 2 PFU (4 × 102 PFU/ml) was obtained with NASBA, compared to 50 PFU (1 × 104PFU/ml) obtained with RT-PCR. No interference was encountered in the amplification of HAV RNA in the presence of excess nontarget RNA or DNA. The NASBA system successfully detected HAV recovered from experimentally inoculated samples of waste water, lettuce, and blueberries. Compared to RT-PCR and other amplification techniques, the NASBA system offers several advantages in terms of sensitivity, rapidity, and simplicity. This technique should be readily adaptable for detection of other RNA viruses in both foods and clinical samples.

scholarly journals Development of a Reverse Transcription-PCR–DNA Enzyme Immunoassay for Detection of “Norwalk-Like” Viruses and Hepatitis A Virus in Stool and Shellfish

2001 ◽  
Vol 67 (2) ◽  
pp. 742-749 ◽  
Author(s):  
Kellogg J. Schwab ◽  
Frederick H. Neill ◽  
Françoise Le Guyader ◽  
Mary K. Estes ◽  
Robert L. Atmar
Keyword(s):  
Enzyme Immunoassay ◽  
Reverse Transcription ◽  
Environmental Samples ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Pcr Amplification ◽  
Rt Pcr ◽  
Reverse Transcription Pcr ◽  
Pcr Product ◽  
Dna Enzyme Immunoassay

ABSTRACT 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.

Effect of Heat Treatment on Hepatitis A Virus and Norovirus in New Zealand Greenshell Mussels (Perna canaliculus)by Quantitative Real-Time Reverse Transcription PCR and Cell Culture

2006 ◽  
Vol 69 (9) ◽  
pp. 2217-2223 ◽  
Author(s):  
JOANNE HEWITT ◽  
GAIL E. GREENING
Keyword(s):  
Cell Culture ◽  
New Zealand ◽  
Reverse Transcription ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Virus Inactivation ◽  
Internal Temperature ◽  
Perna Canaliculus ◽  
Qrt Pcr ◽  
Reverse Transcription Pcr

Quantitative real-time reverse transcription PCR (qRT-PCR) and cell culture (50% tissue culture infectious dose [TCID50]) were used to determine the effect of heat treatments on norovirus and hepatitis A virus (HAV) in the New Zealand Greenshell mussel (Perna canaliculus). Since it is common practice to cook mussels until the shells open, internal temperatures and opening times of mussels on boiling and steaming were determined at regular time intervals. Fifty mussels in batches of six were exposed to boiling and steaming. A mean internal temperature of 90°C (recommended for virus inactivation when maintained for 90 s) was reached after boiling for 170 s, with all 50 mussels open at 210 s. For steaming, the mean internal temperature achieved was only 83°C after 300 s, and all 50 mussels were open. When mussels were steamed for 180 s (mean internal temperature of 63°C), a significant 1.5-log decrease in the HAV titer (log TCID50) was observed. Following the immersion of mussels in boiling water for 180 s (mean internal temperature of 92°C), no viable HAV was detected. For both boiling and steaming experiments, there was no significant change in the norovirus or HAV qRT-PCR titers compared with the controls. Our results show that when New Zealand Greenshell mussels open on heating, their internal temperature may not reach the parameters required for virus inactivation. Immersion for a minimum of 3 min in boiling water rather than steaming is recommended to reduce the risk of viral foodborne illness from contaminated shellfish.

scholarly journals Development, Evaluation, and Standardization of a Real-Time TaqMan Reverse Transcription-PCR Assay for Quantification of Hepatitis A Virus in Clinical and Shellfish Samples

2006 ◽  
Vol 72 (6) ◽  
pp. 3846-3855 ◽  
Author(s):  
M. Isabel Costafreda ◽  
Albert Bosch ◽  
Rosa M. Pint�
Keyword(s):  
Real Time ◽  
Reverse Transcription ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Accurate Estimation ◽  
Acid Extraction ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Reverse Transcription Pcr ◽  
Development Evaluation

ABSTRACT A standardized real-time reverse transcription-PCR (RT-PCR) assay has been developed for an accurate estimation of the number of genome copies of hepatitis A virus (HAV) in clinical and shellfish samples. Real-time procedures were based on the amplification of a fragment of the highly conserved 5′ noncoding region and detection through an internal fluorescent probe, including TaqMan and beacon chemistries, in one- and two-step RT-PCR formats. The best performance in terms of sensitivity and reproducibility was achieved by a one-step TaqMan RT-PCR, with a sensitivity enabling the detection of 0.05 infectious unit and 10 copies of a single-stranded RNA (ssRNA) synthetic transcript. Standard reagents, such as a mengovirus strain and an ssRNA transcript, were employed as controls of nucleic acid extraction and RT-PCR, respectively. The test proved to be highly specific after a broad panel of enteric viruses was tested. Sequence alignment of target regions of the primers and probe proved them to be adequate for the quantification of all HAV genotypes. In addition, a quasispecies analysis of the mutant spectrum indicated that these regions are not prone to variability, thus confirming their robustness.

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