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

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.

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

Canadian Journal of Microbiology ◽

10.1139/w00-134 ◽

2001 ◽

Vol 47 (2) ◽

pp. 153-157 ◽

Cited By ~ 46

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.

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

Applied and Environmental Microbiology ◽

10.1128/aem.64.2.504-508.1998 ◽

1998 ◽

Vol 64 (2) ◽

pp. 504-508 ◽

Cited By ~ 65

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.

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

Water Science & Technology ◽

10.2166/wst.1989.0112 ◽

1989 ◽

Vol 21 (3) ◽

pp. 255-258 ◽

Cited By ~ 4

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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