Enumeration and Differentiation of Vibrio parahaemolyticus and Vibrio vuinificus by DNA-DNA Colony Hybridization Using the Hydrophobic Grid Membrane Filtration Technique for Isolation

1994 ◽  
Vol 57 (2) ◽  
pp. 163-165 ◽  
Author(s):  
CHARLES A. KAYSNER ◽  
CARLOS ABEYTA ◽  
KAREN C. JINNEMAN ◽  
WALTER E. HILL
Keyword(s):  
Crassostrea Gigas ◽  
Vibrio Parahaemolyticus ◽  
Membrane Filtration ◽  
Gene Probe ◽  
Colony Hybridization ◽  
Rapid Technique ◽  
Gene Probes ◽  
Radiolabeled Probe ◽  
Filtration Technique

We have developed a means of differentiating and enumerating Vibrio parahaemolyticus and Vibrio vuinificus by DNA-DNA colony hybridization directly on HGMF filters. V. parahaemolyticus can be detected by a tdh-3-radiolabeled gene probe and V. vuinificus detected by a specific cytotoxin-hemolysin-radiolabeled probe with enumeration directly from autoradiograms. This procedure is more rapid than current techniques allowing enumeration and identification of these two species in samples as diverse as seawater, oyster (Crassostrea gigas), and shrimp (Pandalidae family) within 4 d. Our method is based on a rapid technique (18 h) for isolation and enumeration of V. parahaemolyticus from food using a membrane filtration technique with hydrophobic grid filters (HGMF). With the HGMF method, however, it is not possible to differentiate V. parahaemolyticus from V. vuinificus since on the HGMF-sucrose-based agar used, the two species are indistinguishable as both species are unable to ferment sucrose. Using a combination of the HGMF and selective gene probes, these two species can be differentiated.

scholarly journals Trimethoprim resistance gene inShigella dysenteriae1 isolates obtained from widely scattered locations of Asia

1990 ◽  
Vol 104 (2) ◽  
pp. 219-228 ◽  
Author(s):  
K. Haider ◽  
A. Chatkaeomorakot ◽  
B. A. Kay ◽  
K. A. Talukder ◽  
D. N. Taylor ◽  
...  
Keyword(s):  
Dihydrofolate Reductase ◽  
Plasmid Dna ◽  
The Other ◽  
Shigella Dysenteriae ◽  
Gene Probe ◽  
Type I ◽  
Dhfr Gene ◽  
Colony Hybridization ◽  
Gene Probes ◽  
Resistant Strains

SUMMARYTrimethoprim-resistance genes ofShigella dysenteriae1 strains, isolated from a different location of six different countries of Asia over a 5-year period were characterized by using three different dihydrofolate reductase (DHFR) gene probes. The trimethoprim-resistant (TMPR) strains hybridized only with the type I DHFR gene probe by colony hybridization. None of the strains hybridized with types II and III DHFR gene probes. Southern blot experiments using plasmid DNA extracted from these resistant strains indicated that the type I DHFR genes were either on a 20 MDa plasmid or might be located on the chromosome. None of the other plasmids present inS. dyysenteriae1 strains hybridized with the probe. This indicates that the TMP resistance in theseS. dysenteriae1 strains are mediated by type I DHFR enzyme, and there may be transposition of this type I DHFR gene occurs between the 20 MDa plasmid and the chromosome in this serotype of shigella.

Detection of enteric adenoviruses in south African waters using gene probes

1995 ◽  
Vol 31 (5-6) ◽  
pp. 345-350 ◽  
Author(s):  
B. Genthe ◽  
M. Gericke ◽  
B. Bateman ◽  
N. Mjoli ◽  
R. Kfir
Keyword(s):  
South African ◽  
Water Samples ◽  
Cellulose Fibre ◽  
Gene Probe ◽  
Viral Dna ◽  
Virus Particles ◽  
Gene Probes ◽  
Probe Hybridization ◽  
Enteric Adenoviruses ◽  
Effect Of Chlorine

Gene probes developed locally for both enteric Adenoviruses 40 and 41 were used to determine whether these viruses were present in both raw and treated waters. Approximately sixty water samples were concentrated by ultrafiltration and analysed directly for the presence of enteric adenoviruses. Three pretreatment techniques, namely sephadex columns, cellulose fibre and GenecleanTM were tested for the removal of inhibitory substances from concentrated water samples. The effect of chlorine treatment on viral detection using gene probe hybridization was also examined by exposing adenoviruses to chlorine concentrations of up to 20mg/l for 1 hour. Enteric adenoviruses were detected in up to 59% of both raw and treated waters analysed. Cellulose fibre and GenecleanTM were found to successfully remove inhibitory substances from concentrated raw waters. Viral DNA was detected after exposure to a range of chlorine concentrations indicating that the viruses detected in the treated waters may have been inactivated virus particles.

Use of gene probes for the detection of quiescent enteric bacteria in marine and fresh waters

1995 ◽  
Vol 31 (5-6) ◽  
pp. 291-298
Author(s):  
Sally A. Anderson ◽  
Gillian D. Lewis ◽  
Michael N. Pearson
Keyword(s):  
Membrane Filtration ◽  
Enteric Bacteria ◽  
Probe Method ◽  
Detection Methods ◽  
23S Rrna ◽  
Specific Gene ◽  
Gene Probe ◽  
Selective Media ◽  
Selective Enrichment ◽  
E Coli

Specific gene probe detection methods that utilise a non-selective culturing step were tested for the ability to recognise the presence of quiescent enteric bacteria (Escherichia coli and Enterococcus faecalis ) within illuminated freshwater and seawater microcosms. An E. coli specific uidA gene probe and a 23S rRNA oligonucleotide probe for Enterococci were compared with recoveries using membrane filtration and incubation on selective media (mTEC and mE respectively). From these microcosm experiments a greater initial detection (from 4 hours to 1 day) of E. coli and Ent. faecalis using gene probe methods was observed. Additionally, a comparison of E. coli direct viable counts (DVC) in sunlight exposed microcosms with recoveries by selective media and gene probe methods revealed a large number of viable non-culturable cells. This suggests that enumeration of E. coli by a gene probe method is limited by the replication of the bacteria during the initial non-selective enrichment step. The detection of stressed Ent. faecalis by the oligonucleotide gene probe method was significantly greater than recovery on selective mE agar, indicating an Enterococci non-growth phase.

The Influence of Carbohydrate Gelling Agents on Rat Intestinal Transport of Monosaccharides and Neutral Amino Acids in Vitro

1980 ◽  
Vol 59 (5) ◽  
pp. 373-380 ◽  
Author(s):  
B. Elsenhans ◽  
U. Süfke ◽  
R. Blume ◽  
W. F. Caspary
Keyword(s):  
Intestinal Absorption ◽  
Membrane Filtration ◽  
Intestinal Transport ◽  
Michaelis Constant ◽  
Experimental Conditions ◽  
Plant Polysaccharides ◽  
Gelling Agents ◽  
Layer Resistance ◽  
Filtration Technique

1. In the present investigation with rings of everted rat small intestine, carbohydrate gelling agents (plant polysaccharides) such as guaran, pectin, tragacanth, carubin and carrageenan were employed to study their direct effect on intestinal absorption of α-methyl-d-glucoside, d-galactose, l-leucine and l-phenylalanine. 2. Inhibition was found to correlate with the viscosity of the incubation medium, a function only of the polysaccharide concentration, and was independent of other properties of the carbohydrate gelling agents. 3. Reversal of this inhibition was achieved either by washing the tissue free of polysaccharide or by raising tissue agitation. 4. Uptake kinetics in polysaccharide-containing solutions revealed a marked increase of the apparent Michaelis constant although the maximal transport capacity remained essentially unaltered. 5. Since there was no binding of the substrate by the polysaccharides under experimental conditions as judged by a membrane filtration technique, it is concluded that carbohydrate gelling agents may impair intestinal absorption by means of an increased unstirred layer resistance.

Real-Time PCR Quantification of Vibrio parahaemolyticus in Oysters Using an Alternative Matrix

2004 ◽  
Vol 67 (11) ◽  
pp. 2424-2429 ◽  
Author(s):  
G. E. KAUFMAN ◽  
G. M. BLACKSTONE ◽  
M. C. L. VICKERY ◽  
A. K. BEJ ◽  
J. BOWERS ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Vibrio Parahaemolyticus ◽  
Pcr Amplification ◽  
Oyster Tissue ◽  
Pcr Assay ◽  
Colony Hybridization ◽  
The Real ◽  
Mantle Fluid ◽  
June July

This study examined the relationship between levels of total Vibrio parahaemolyticus found in oyster tissues and mantle fluid with the goal of using mantle fluid as a template matrix in a new quantitative real-time PCR assay targeting the thermolabile hemolysin (tlh) gene for the enumeration of total V. parahaemolyticus in oysters. Oysters were collected near Mobile Bay, Ala., in June, July, and September and tested immediately after collection and storage at 26°C for 24 h. Initial experiments using DNA colony hybridization targeting tlh demonstrated that natural V. parahaemolyticus levels in the mantle fluid of individual oysters were strongly correlated (r = 0.85, P < 0.05) with the levels found in their tissues. When known quantities of cultured V. parahaemolyticus cells were added to real-time PCR reactions that contained mantle fluid and oyster tissue matrices separately pooled from multiple oysters, a strong linear correlation was observed between the real-time PCR cycle threshold and the log concentration of cells inoculated into each PCR reaction (mantle fluid: r = 0.98, P < 0.05; and oyster: r = 0.99, P < 0.05). However, the mantle fluid exhibited less inhibition of the PCR amplification than the homogenized oyster tissue. Analysis of natural V. parahaemolyticus populations in mantle fluids using both colony hybridization and real-time PCR demonstrated a significant (P < 0.05) but reduced correlation (r =−0.48) between the two methods. Reductions in the efficiency of the real-time PCR that resulted from low population densities of V. parahaemolyticus and PCR inhibitors present in the mantle fluid of some oysters (with significant oyster-to-oyster variation) contributed to the reduction in correlation between the methods that was observed when testing natural V. parahaemolyticus populations. The V. parahaemolyticus–specific real-time PCR assay used for this study could estimate elevated V. parahaemolyticus levels in oyster mantle fluid within 1 h from sampling time.

Recovery of feline calicivirus infectious particles and genome from water: comparison of two concentration techniques

2003 ◽  
Vol 47 (3) ◽  
pp. 97-101 ◽  
Author(s):  
B. Gassilloud ◽  
M. Duval ◽  
L. Schwartzbrod ◽  
C. Gantzer
Keyword(s):  
Recovery Rate ◽  
Membrane Filtration ◽  
Glass Wool ◽  
Feline Calicivirus ◽  
Concentration Step ◽  
Filtration Technique

The aim of this work was to determine the recovery rate of feline calicivirus (FCV-F9) infectious particles and genome from water after a concentration step using either adsorption elution on glass wool or filtration through an electropositive membrane. The results showed that the membrane filtration technique allowed a 75% recovery rate of FCV-F9 infectious particles while the yield was only 5.3% for FCV-F9 genome. Using the glass wool adsorption-elution technique, the recovery rate of FCV-F9 infectious particles was 0.5% while the yield was 102.5% with Poliovirus 1.

Effects of Flash Freezing, Followed by Frozen Storage, on Reducing Vibrio parahaemolyticus in Pacific Raw Oysters (Crassostrea gigas)

2009 ◽  
Vol 72 (1) ◽  
pp. 174-177 ◽  
Author(s):  
CHENGCHU LIU ◽  
JIANZHANG LU ◽  
YI-CHENG SU
Keyword(s):  
Crassostrea Gigas ◽  
Vibrio Parahaemolyticus ◽  
Vibrio Vulnificus ◽  
Frozen Storage ◽  
Most Probable Number ◽  
Freezing Process ◽  
Probable Number ◽  
Pacific Oysters ◽  
Subsequent Storage ◽  
Shellfish Sanitation

This study investigated the effects of flash freezing, followed by frozen storage, on reducing Vibrio parahaemolyticus in Pacific raw oysters. Raw Pacific oysters were inoculated with a five-strain cocktail of V. parahaemolyticus at a total level of approximately 3.5 × 105 most probable number (MPN) per gram. Inoculated oysters were subjected to an ultralow flash-freezing process (−95.5°C for 12 min) and stored at −10, −20, and −30°C for 6 months. Populations of V. parahaemolyticus in the oysters declined slightly by 0.22 log MPN/g after the freezing process. Subsequent storage of frozen oysters at −10, −20, and −30°C resulted in considerable reductions of V. parahaemolyticus in the oysters. Storing oysters at −10°C was more effective in inactivating V. parahaemolyticus than was storage at −20 or −30°C. Populations of V. parahaemolyticus in the oysters declined by 2.45, 1.71, and 1.45 log MPN/g after 1 month of storage at −10, −20, and −30°C, respectively, and continued to decline during the storage. The levels of V. parahaemolyticus in oysters were reduced by 4.55, 4.13, and 2.53 log MPN/g after 6 months of storage at −10, −20, and −30°C, respectively. Three process validations, each separated by 1 week and conducted according to the National Shellfish Sanitation Program's postharvest processing validation–verification interim guidance for Vibrio vulnificus and Vibrio parahaemolyticus, confirmed that a process of flash freezing, followed by storage at −21 ± 2°C for 5 months, was capable of achieving greater than 3.52-log (MPN/g) reductions of V. parahaemolyticus in half-shell Pacific oysters.

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