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.

Development of PCR Methods for Enteric Virus Detection in Water

1993 ◽  
Vol 27 (3-4) ◽  
pp. 211-218 ◽  
Author(s):  
K. J. Schwab ◽  
R. De Leon ◽  
M. D. Sobsey
Keyword(s):  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Detection System ◽  
Virus Detection ◽  
Enteric Virus ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Sample Concentration ◽  
Spin Column ◽  
Polymerase Chain

This study developed a reverse transcriptase-polymerase chain reaction (RT-PCR) detection system for enteric viruses in sample concentrates obtained by conventional filter adsorption-elution methods. One liter beef extract (BE)-glycine (G) eluant seeded with poliovirus 1 and hepatitis A virus (HAV) was used as a model system and the eluant further processed for RT-PCR compatibility. Sample concentration and purification procedures consisted of polyethylene glycol (PEG) precipitation, Pro-Cipitate (Affinity Technology, New Brunswick, NJ) precipitation, a second PEG precipitation, spin-column chromatography, and ultrafiltration. Sample volumes are reduced from 1 L to 20-40 µL and purified sufficiently for viral detection by RT-PCR. As little as 3 PFU of poliovirus 1 in an initial 1 L eluate were detected by RT-PCR.

Rapid PCR-based monitoring of infectious enteroviruses in drinking water

1997 ◽  
Vol 35 (11-12) ◽  
pp. 423-427 ◽  
Author(s):  
K. S. Reynolds ◽  
C. P. Gerba ◽  
I. L. Pepper
Keyword(s):  
Cell Culture ◽  
Hepatitis A Virus ◽  
Virus Detection ◽  
Integrated Approach ◽  
Rt Pcr ◽  
Direct Pcr ◽  
Integrated Method ◽  
Cell Culture Assay ◽  
Conventional Cell ◽  
Reaction Volumes

Currently, the standard method for the detection of enteroviruses and hepatitis A virus in water involves cell culture assay which is expensive and time consuming. Direct RT-PCR offers a rapid and sensitive alternative to virus detection but sensitivity is often reduced by PCR inhibitory substances and the requirement for small reaction volumes. Rapid methods for detection of infectious enteroviruses in PCR inhibitory environmental samples are being developed utilising an integrated cell culture/PCR approach (ICC/PCR). With this approach, 300–4001 of water were concentrated using charged filters followed by a modified 11, 1.5% BEV/glycine elution and organic flocculation reconcentration. Water concentrates were analysed by direct RT-PCR, conventional cell culture and ICC/PCR. For ICC/PCR, sample concentrates were incubated with BGM or FRhK cells for 24–48h. The cell culture lysates were collected following freeze-thaw cycles, centrifuged, resin column purified and PCR amplified. In this study viruses known to be present by cell culture analysis could not be detected by direct PCR. Using the integrated method, virus concentrations as low as 0.001MPN/l of original water were detected in samples which were previously inhibitory to direct PCR. In addition, confirmed enterovirus results were achieved as soon as 48h against 5–16d with cell culture alone. Therefore, the integrated approach overcame some of the traditional problems associated with conventional cell culture and direct RT-PCR by allowing rapid, confirmed detection of low levels of enteroviruses in PCR inhibitory samples.

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.

Virucidal effect of UV light on hepatitis a virus in sea water: evaluation with cell culture and RT-PCR

1995 ◽  
Vol 31 (5-6) ◽  
pp. 157-160 ◽  
Author(s):  
F. Lévêque ◽  
J. M. Crance ◽  
C. Beril ◽  
L. Schwartzbrod
Keyword(s):  
Hepatitis A ◽  
Infectious Virus ◽  
Sea Water ◽  
Hepatitis A Virus ◽  
Uv Light ◽  
Contaminated Water ◽  
Rt Pcr ◽  
Genomic Amplification ◽  
Virucidal Effect ◽  
Polymerase Chain

Virucidal effect of UV light on hepatitis A virus was investigated in artificial sea water. Infectious virus was no longer detectable after 15 min irradiation of 3 liter experimentally contaminated water. Genomic amplification by polymerase chain reaction after reverse transcription allowed the detection of viral RNA in all samples even after 60 min irradiation.

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.

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.

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.

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