Comparison of Poliovirus Detection in Water by Cell Culture and Nucleic Acid Hybridization

1993 ◽  
Vol 27 (3-4) ◽  
pp. 315-319
Author(s):  
Carlos E. Enriquez ◽  
Morteza Abbaszadegan ◽  
Ian L. Pepper ◽  
Kenneth J. Richardson ◽  
Aaron B. Margolin ◽  
...  
Keyword(s):  
Cell Culture ◽  
Tissue Culture ◽  
Nucleic Acid ◽  
Water Samples ◽  
Environmental Water ◽  
Nucleic Acid Hybridization ◽  
Well Water ◽  
Gene Probe ◽  
Hybridization Technique ◽  
Water Filter

The nucleic acid hybridization technique has been used to detect viral nucleic acid in environmental water samples. This type of assay, in contrast with tissue culture assays, may not distinguish between viable and non-viable viruses. We evaluated, by comparison with tissue culture infectivity assay (plaque forming method), the ability of the gene probe assay to detect viable poliovirus 1 (LSc) in well water, autoclaved well water, filter-sterilized well water and autoclaved phosphate buffered saline kept at 37° C and 15° C for 75 days, and in dechlorinated tapwater held at room temperature. A gradual decline in numbers of poliovirus was observed in all of the samples by cell culture assay. With the exception of autoclaved well water and phosphate buffer samples, a parallel decline in virus detectable by gene probe occurred in all other water samples.

Comparison of Cell Culture and a Poliovirus Gene Probe Assay for the Detection of Enteroviruses in Environmental Water Samples

1993 ◽  
Vol 27 (3-4) ◽  
pp. 311-314 ◽  
Author(s):  
Aaron B. Margolin ◽  
Charles P. Gerba ◽  
Kenneth J. Richardson ◽  
Jaime E. Naranjo
Keyword(s):  
Cell Culture ◽  
Activated Sludge ◽  
Water Samples ◽  
Cdna Probe ◽  
Culture Method ◽  
Tidal River ◽  
Nucleic Acid Hybridization ◽  
Treated Wastewater ◽  
Gene Probe ◽  
Naturally Occurring

Nucleic acid hybridization provides a rapid non-cell culture method for the detection of enteric viruses in water. The purpose of this work was to compare the detection of naturally occurring enteroviruses by cell culture with their detection by a poliovirus gene probe in various types of water samples. Samples of activated sludge effluent, tertiary treated wastewater (activated sludge, filtration and passage through reverse osmosis), ground water, surface water and tidal river water were processed through 1 MDS Virozorb filters to concentrate any naturally occurring virus. Viruses were eluted from the filters with pH 9.5 beef extract and reduced in volume by flocculation to 20-30 ml. These concentrates were then assayed in the BGM cell line by the cytopathogenic effects (CPE) method and by a poliovirus cDNA probe (base pairs 115-7440) labeled with 32P. A total of 233 samples were assayed in this manner. In slightly more than 93% of the samples gene probe and cell culture yielded the same results. Of these samples 36 were positive by gene probe and 28 by cell culture assay. Positive samples for gene probe were confirmed by treatment with NaOH or RNAse and then reprobed. Samples demonstrating CPE upon primary passage were confirmed positive by subsequent passage of cell lysate on a new monolayer of BGM cells. Ten samples were positive by gene probe and negative by cell culture, and 4 samples were negative by gene probe and positive by cell culture.

Detection and identification of poliovirus in environmental samples using nucleic acid hybridization

1990 ◽  
Vol 36 (9) ◽  
pp. 664-669 ◽  
Author(s):  
David R. Preston ◽  
G. Rasul Chaudhry ◽  
Samuel R. Farrah
Keyword(s):  
Tissue Culture ◽  
Nucleic Acid ◽  
Environmental Samples ◽  
Nucleic Acid Hybridization ◽  
Dot Blot ◽  
Polio Virus ◽  
Detection And Identification ◽  
Specificity And Sensitivity ◽  
Hybridization Procedure ◽  
Great Specificity

A procedure was developed to effectively extract viral RNA from poliovirus tissue-culture lysates while eliminating the hybridization background associated with tissue cultures uninfected with poliovirus. Poliovirus cDNA cloned into a pUC vector was used as probe. Both the recombinant plasmids and the cDNA showed great specificity towards poliovirus. However, both probes hybridized with the single-stranded DNA coliphage [Formula: see text]. Tissue culture was found to be an effective method to increase the number of viruses found in environmental samples to a level detectable by hybridization procedures, whereas direct hybridization of RNA from unamplified and highly concentrated raw wastewater showed poor hybridization signals. The specificity and sensitivity of the hybridization procedure developed during these studies indicate that this method may be best suited for the identification rather than the detection of viruses isolated from environmental samples. Key words: nucleic acid hybridization, polio virus, water, dot blot.

Characterization of the diversity of sulfate-reducing bacteria in soil and mining waste water environments by nucleic acid hybridization techniques

1994 ◽  
Vol 40 (11) ◽  
pp. 955-964 ◽  
Author(s):  
Anita J. Telang ◽  
Gerrit Voordouw ◽  
Sara Ebert ◽  
Neili Sifeldeen ◽  
Julia M. Foght ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Bacterial Communities ◽  
Genomic Dna ◽  
Sulfate Reducing Bacteria ◽  
Oil Field ◽  
Nucleic Acid Hybridization ◽  
Gene Probe ◽  
Sulfate Reducing ◽  
Reducing Bacteria ◽  
Field Production

Nucleic acid hybridization techniques were used to characterize the sulfate-reducing bacterial communities at seven waste water and two soil sites in Canada. Genomic DNA was obtained from liquid enrichment cultures of samples taken from these nine sites. The liquid enrichment protocol favored growth of the sulfate-reducing bacterial component of the communities at these sites. The genomic DNA preparations were analyzed with (i) a specific gene probe aimed at a single genus (Desulfovibrio), (ii) a general 16S rRNA gene probe aimed at all genera of sulfate-reducing bacteria and other bacteria, and (iii) whole genome probes aimed at specific bacteria. This three-pronged approach provided information on the sulfate-reducing bacterial community structures for the nine sites. These were compared with each other and with the sulfate-reducing bacterial communities of western Canadian oil field production waters, studied previously. It was found that there is considerable diversity in the sulfate-reducing bacterial community at each site. Most sulfate-reducing bacteria isolated from distinct sites are genomically different and differ also from sulfate-reducing bacteria found in oil field production waters.Key words: sulfate-reducing bacteria, genomic diversity, nucleic acid hybridization, microbial community.

scholarly journals Comparison of Techniques for Detection of Barley Yellow Dwarf Virus-PAV-IL

Plant Disease ◽  
1997 ◽  
Vol 81 (11) ◽  
pp. 1236-1240 ◽  
Author(s):  
Antonia R. Figueira ◽  
Leslie L. Domier ◽  
Cleora J. D'Arcy
Keyword(s):  
Nucleic Acid ◽  
Barley Yellow Dwarf Virus ◽  
Infected Tissue ◽  
Yellow Dwarf ◽  
Nucleic Acid Hybridization ◽  
Dwarf Virus ◽  
Enzyme Linked Immunosorbent Assay ◽  
Hybridization Technique ◽  
Barley Yellow Dwarf ◽  
Yellow Dwarf Virus

Detection of barley yellow dwarf virus (BYDV)-PAV-IL by an improved nucleic acid hybridization technique, using a nonradioactive probe with chromogenic and chemiluminescent substrates, was compared with detection by polymerase chain reaction (PCR), double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) with polyclonal antibodies, and triple antibody sandwich ELISA with polyclonal and monoclonal antibodies. Each method was used to detect purified virus and virus in sap extracts from infected oat leaves. The detection limits for both ELISA procedures were 1 ng of purified BYDV-PAV-IL and the equivalent of 78 ng of infected tissue. Nucleic acid hybridization with either chemiluminescent or chromogenic substrates also detected as little as 1 ng of purified BYDV-PAV-IL, but it was slightly more sensitive at detecting virus in tissue extracts (25 ng of infected tissue). The most sensitive detection technique was PCR amplification, which could detect as little as 0.1 pg of RNA extracted from purified virus and detected viral RNA in the equivalent of 0.5 pg of infected leaf tissue.

scholarly journals Detection of Vibrio cholerae by Real-Time Nucleic Acid Sequence-Based Amplification

2007 ◽  
Vol 73 (5) ◽  
pp. 1457-1466 ◽  
Author(s):  
Else M. Fykse ◽  
Gunnar Skogan ◽  
William Davies ◽  
Jaran Strand Olsen ◽  
Janet M. Blatny
Keyword(s):  
Nucleic Acid ◽  
Vibrio Cholerae ◽  
Real Time ◽  
Water Samples ◽  
Environmental Water ◽  
Nucleic Acid Sequence ◽  
Environmental Water Samples ◽  
Dnase Treatment ◽  
Nasba Assay ◽  
Toxigenic Strains

ABSTRACT A multitarget molecular beacon-based real-time nucleic acid sequence-based amplification (NASBA) assay for the specific detection of Vibrio cholerae has been developed. The genes encoding the cholera toxin (ctxA), the toxin-coregulated pilus (tcpA; colonization factor), the ctxA toxin regulator (toxR), hemolysin (hlyA), and the 60-kDa chaperonin product (groEL) were selected as target sequences for detection. The beacons for the five different genetic targets were evaluated by serial dilution of RNA from V. cholerae cells. RNase treatment of the nucleic acids eliminated all NASBA, whereas DNase treatment had no effect, showing that RNA and not DNA was amplified. The specificity of the assay was investigated by testing several isolates of V. cholerae, other Vibrio species, and Bacillus cereus, Salmonella enterica, and Escherichia coli strains. The toxR, groEL, and hlyA beacons identified all V. cholerae isolates, whereas the ctxA and tcpA beacons identified the O1 toxigenic clinical isolates. The NASBA assay detected V. cholerae at 50 CFU/ml by using the general marker groEL and tcpA that specifically indicates toxigenic strains. A correlation between cell viability and NASBA was demonstrated for the ctxA, toxR, and hlyA targets. RNA isolated from different environmental water samples spiked with V. cholerae was specifically detected by NASBA. These results indicate that NASBA can be used in the rapid detection of V. cholerae from various environmental water samples. This method has a strong potential for detecting toxigenic strains by using the tcpA and ctxA markers. The entire assay including RNA extraction and NASBA was completed within 3 h.

scholarly journals Optimizing a Suspension Culture Method with a Decreased Cost to Detect Enteroviruses in Water to Increase Surveillance Access

2020 ◽  
Vol 11 (2) ◽  
pp. 35-44
Author(s):  
Stephanie Tornberg-Belanger ◽  
Jonathan Sreter ◽  
Aaron Margolin
Keyword(s):  
Cell Culture ◽  
Nucleic Acid ◽  
Water Samples ◽  
Financial Burden ◽  
Virus Detection ◽  
Culture Method ◽  
Virus Concentration ◽  
Viral Nucleic Acid ◽  
A Cell ◽  
Materials Used

Enteroviruses are a public health threat due to the high incidence of infections and potential for serious illness or death. Some laboratories in high-income countries detect enteroviruses in water by integrating cell culture and PCR (ICC/PCR). This combined method carries a high financial burden, due in part to specialized cell culture equipment. Therefore, we expanded upon a pilot study to reduce the cost by using common laboratory polypropylene tubes to create a cell culture in suspension. We optimized the protocol by determining minimal incubation periods post-infection as a function of the initial virus concentration. Cells in suspension and traditional monolayers were inoculated with poliovirus and incubated in 8-hour intervals up to 48 hours prior to extraction. Quantitative PCR (qPCR) was used to detect viral nucleic acid targets. Treated and raw water samples were seeded with virus and the suspension ICC/qPCR protocol used to ascertain whether the protocol performed similar to directly seeding cells. No variation in virus detection occurred using the suspension ICC/qPCR or monolayer ICC/qPCR (p = 0.95). In surface water samples, viral nucleic acid was successfully detected, with no significant increase after 32 h (p > 0.05). Suspension ICC/qPCR is as effective as monolayer ICC/qPCR in detecting enteroviruses in surface waters. Materials used in the suspension ICC/qPCR have a lower monetary cost than traditional cell culture materials without loss of sensitivity. More accessible testing of waters for enterovirus contamination through cost reduction has the potential to reduce human exposure and disease.

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