scholarly journals Immunomagnetic Capture PCR for Rapid Concentration and Detection of Hepatitis A Virus from Environmental Samples

1998 ◽  
Vol 64 (2) ◽  
pp. 504-508 ◽  
Author(s):  
N. Jothikumar ◽  
Dean O. Cliver ◽  
Tadesse W. Mariam
Keyword(s):  
Cell Culture ◽  
Phosphate Buffer ◽  
Field Trial ◽  
Environmental Samples ◽  
Hepatitis A ◽  
Magnetic Beads ◽  
Hepatitis A Virus ◽  
Membrane Filter ◽  
Rt Pcr ◽  
Immunomagnetic Capture

ABSTRACT We studied the concentration of hepatitis A virus (HAV) from environmental samples by membrane filter-based urea-arginine phosphate buffer and its detection by using immunomagnetic capture (IC) reverse transcription (RT)-PCR (IC PCR). Magnetic beads coated with anti-HAV rabbit antibodies were used for enrichment and concentration of HAV from environmental samples. IC PCR is sensitive enough to detect as few as 0.04 PFU of cell culture-adapted HAV in inoculated water and sewage samples. IC PCR is specific and does not yield positive reactions with poliovirus 1, HAV RNA, or selected bacteriophages. IC concentrates viruses suspended in small volumes to microliter volumes that can be used directly in RT-PCR. IC concentration of viruses from sewage samples without concentration of inhibitory substances is important for successful RT-PCR detection. In a field trial, 2 of 18 raw sewage samples tested by IC PCR were 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.

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.

Application of the PCR Technique to the Detection of Hepatitis a Virus in the Environment

1993 ◽  
Vol 27 (3-4) ◽  
pp. 223-225 ◽  
Author(s):  
M. Divizia ◽  
G. Morace ◽  
R. Gabrieli ◽  
G. Pisani ◽  
A. Panà
Keyword(s):  
Cell Culture ◽  
Environmental Samples ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Elisa Method ◽  
Traditional Methods ◽  
Three Samples ◽  
Hybridization Test ◽  
Pcr Method ◽  
The Difference

Rapid, sensitive and specific teciiniques are needed to detect Hepatitis A virus in environmental samples. We have applied the method of Jansen et al. to concentrate river samples. In the analysis of 13 samples only two proved to be positive using the Elisa method (15.3%). Three samples were positive on cell culture (23%). The hybridization test resulted in 38.4% positive samples compared to the 67% obtained using the PCR method. The PCR showed the highest sensibility when compared to the traditional methods (cell culture and Elisa) and the differences were statistically significant. The difference between hybridization test and PCR was not statistically significant.

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.

scholarly journals Combined Immunomagnetic Separation-Molecular Beacon-Reverse Transcription-PCR Assay for Detection of Hepatitis A Virus from Environmental Samples

2004 ◽  
Vol 70 (7) ◽  
pp. 4371-4374 ◽  
Author(s):  
Khaled H. Abd El Galil ◽  
M. A. El Sokkary ◽  
S. M. Kheira ◽  
Andre M. Salazar ◽  
Marylynn V. Yates ◽  
...  
Keyword(s):  
Real Time ◽  
Reverse Transcription ◽  
Environmental Samples ◽  
Hepatitis A ◽  
Molecular Beacon ◽  
Hepatitis A Virus ◽  
Immunomagnetic Separation ◽  
Rt Pcr ◽  
Pcr Assay ◽  
The Real

ABSTRACT In this study, a molecular-beacon-based real-time reverse transcription (RT)-PCR assay was developed to detect the presence of hepatitis A virus (HAV) in environmental samples. A 125-bp, highly conserved 5′ noncoding region of HAV was targeted. The sensitivity of the real-time RT-PCR assay was tested with 10-fold dilutions of viral RNA, and a detection limit of 1 PFU was obtained. The specificity of the assay was demonstrated by testing with other environmental pathogens and indicator microorganisms, and only HAV was positively identified. When combined with immunomagnetic separation, the real-time RT-PCR assay successfully detected as few as 20 PFU in seeded groundwater samples. Because of its simplicity and specificity, this assay has broad applications for the rapid detection of HAV in contaminated foods or water.

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.

scholarly journals A Strategy for Detection of Viruses in Groundwater by PCR

1999 ◽  
Vol 65 (2) ◽  
pp. 444-449 ◽  
Author(s):  
Morteza Abbaszadegan ◽  
Peter Stewart ◽  
Mark LeChevallier
Keyword(s):  
Cell Culture ◽  
Large Volume ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Processing Technique ◽  
Molecular Techniques ◽  
Pcr Analysis ◽  
Rt Pcr ◽  
Sample Processing ◽  
Specific Primers

ABSTRACT We evaluated the use of the PCR for detection of enteric viruses in groundwater. To do this, we used an improved sample-processing technique and a large-volume amplification protocol. The objective of this study was to use advanced molecular techniques to develop a rapid and simple method which can be used by the water industry for detection of viral contamination in a variety of water samples. The strategy described here fulfills the water industry’s need for a rapid, reliable, easily performed method for analyzing groundwater for virus contamination. Viruses were detected after concentration from at least 400 gallons (1,512 liters) of water by a filter adsorption and elution method, which resulted in a concentrate containing viruses. A total of 150 samples were analyzed by performing cell culture assays for enteroviruses and by performing reverse transcription PCR (RT-PCR) analyses for enteroviruses, hepatitis A virus, and rotavirus. Thirteen samples (8.7%) produced cellular cytopathic effects when the Buffalo green monkey cell line was used. When primers specific for enteroviruses were used in RT-PCR, 40 of 133 samples (30.1%) tested positive for the presence of enterovirus RNA. When hepatitis A virus-specific primers were used, 12 of 139 samples (8.6%) were considered positive for the presence of hepatitis A viral RNA. The RT-PCR analysis performed with rotavirus-specific primers identified 18 of 130 samples (13.8%) that were positive for rotavirus RNA sequences. Our sample-processing technique and large-volume PCR protocol (reaction volume, 300 μl) resulted in sufficient removal or dilution of inhibitors so that more than 95% of the samples could be assayed by PCR. Because of its sensitivity for detecting viral nucleic acid sequences, PCR analysis should produce more positive results than cell culture analysis. Since either cell culture analysis or PCR can reveal only a “snapshot” of the quality of the groundwater being sampled, PCR seems to be a desirable rapid initial screening tool.

Hepatitis a Virus Concentration from Experimentally Contaminated Distilled, Tap, Waste and Seawater

1989 ◽  
Vol 21 (3) ◽  
pp. 255-258 ◽  
Author(s):  
Evangélos Biziagos ◽  
Jacques Passagot ◽  
Jean-Marc Crance ◽  
Robert Deloince
Keyword(s):  
Cell Culture ◽  
Polyethylene Glycol ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Tap Water ◽  
Virus Concentration ◽  
Beef Extract ◽  
Polyethylene Glycol 6000

The concentration of cell-culture-adapted hepatitis A virus (HAV) from experimentally contaminated distilled, drinking, waste and seawater was performed by using a filter adsorption-elu-tion method in the following conditions: HAV seeded in water was adsorbed at pH 4.0 to two nitrocellulose membranes (1.2 and 0.45 µm porosity for distilled and tap water or 8.0 and 3.0 µm porosity for waste and seawater), then eluted by 3% beef-extract at pH 8.5 and further concentrated by polyethylene glycol 6000 precipitation. Thus, HAV in 5 to 50 liters of seeded waters was concentrated approximately 1,700 to 17,000 fold with greater than 70% recovery of the initial virus added to the samples.

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