Detection of toxigenic<i>Vibrio cholerae</i>from environmental water samples by an enrichment broth cultivation-pit-stop semi-nested PCR procedure

Theron, Jacques; Cilliers, Jakkie; Preez, Martella du; Brözel, Volker S.; Venter, Stephanus

doi:10.1046/j.1365-2672.2000.01140.x

Cited by 48 publications

(31 citation statements)

References 44 publications

(86 reference statements)

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“…This was a significant improvement over the previously described SYBR green I assay, which had a detection limit of 10 3 CFU per reaction (10). Several groups have similarly employed a DNA extraction step prior to PCR analysis of environmental water samples for V. cholerae (12,15,16,23,27). In comparison, a major advantage of the DNA extraction method used in this study is that it can be easily adapted to filter very large volumes of water.…”

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confidence: 95%

Molecular-Beacon Multiplex Real-Time PCR Assay for Detection of Vibrio cholerae

Gubala

Proll

2006

Appl Environ Microbiol

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mentioning

confidence: 95%

Molecular-Beacon Multiplex Real-Time PCR Assay for Detection of Vibrio cholerae

Gubala

Proll

2006

Appl Environ Microbiol

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“…This may explain the failure of traditional culture techniques to isolate this organism from contaminated water and food samples implicated in food-borne outbreaks (10,27,47). Several investigators have developed PCR and DNA probe techniques for the detection of pathogenic Vibrio species (7,12,14,19,23,25,40,41,44,46; S. C. Arya, Letter, J. Clin. Microbiol.…”

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confidence: 99%

TaqMan PCR for Detection of Vibrio cholerae O1, O139, Non-O1, and Non-O139 in Pure Cultures, Raw Oysters, and Synthetic Seawater

Lyon

2001

Appl Environ Microbiol

116

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Vibrio cholerae is recognized as a leading human waterborne pathogen. Traditional diagnostic testing for Vibrio is not always reliable, because this bacterium can enter a viable but nonculturable state. Therefore, nucleic acid-based tests have emerged as a useful alternative to traditional enrichment testing. In this article, a TaqMan PCR assay is presented for quantitative detection of V. cholerae in pure cultures, oysters, and synthetic seawater. Primers and probe were designed from the nonclassical hemolysin (hlyA) sequence of V. cholerae strains. This probe was applied to DNA from 60 bacterial strains comprising 21 genera. The TaqMan PCR assay was positive for all of the strains of V. cholerae tested and negative for all other species of Vibrio tested. In addition, none of the other genera tested was amplified with the TaqMan primers and probe used in this study. The results of the TaqMan PCR with raw oysters and spiked with V. cholerae serotypes O1 and O139 were comparable to those of pure cultures. The sensitivity of the assay was in the range of 6 to 8 CFU g ؊1 and 10 CFU ml ؊1 in spiked raw oyster and synthetic seawater samples, respectively. The total assay could be completed in 3 h. Quantification of the Vibrio cells was linear over at least 6 log units. The TaqMan probe and primer set developed in this study can be used as a rapid screening tool for the presence of V. cholerae in oysters and seawater without prior isolation and characterization of the bacteria by traditional microbiological methods.Vibrio cholerae is a waterborne pathogen that causes gastrointestinal disorders with a wide range of clinical manifestations, including vomiting and rice-like diarrhea (24). The association of human illness with consumption of V. choleraecontaminated oysters, seawater, and other shellfish is well documented (29, 37). Consumption of raw oysters correlates strongly with gastrointestinal infections, and several Vibrio species, including strains of Vibrio parahaemolyticus, Vibrio vulnificus, and V. cholerae, have been implicated as the causative agents.Coastal areas with brackish waters and estuarine regions are niches for many Vibrio species, including strains of toxigenic O1 V. cholerae. Epidemic cholera strains are endemic in several regions, including the U.S. gulf coast and Australia, and are occasionally involved in illnesses in these regions (24). Because Vibrio species attach to material suspended in water, shellfish and mollusks that are in these environments can be expected to consume Vibrio during feeding (24).Traditional identification methods currently used are time-consuming and laborious, requiring prolonged incubation and selective enrichment to reduce the growth of background flora. Vibrio cells may also enter a viable but not culturable (VBNC) state, caused by nutrient starvation and physical stress. This may explain the failure of traditional culture techniques to isolate this organism from contaminated water and food samples implicated in food-borne outbreaks (10,27,47). Several investigator...

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“…The detection sensitivity was lower in seeded tap water samples, probably due to the stress posed by chlorinating agents in the potable water. In earlier studies also, sensitivity of PCR was lower in tap water samples 14,15 . Molecular monitoring of microbial ecosystems is a very rapid and reliable tool to detect specifi c micro-organisms.…”

Section: Resultsmentioning

confidence: 69%

Semi-nested polymerase chain reaction for detection of toxigenic Vibrio cholerae from environmental water samples

et al. 2007

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A rapid and sensitive direct cell semi-nested PCR assay was developed for the detection of viable toxigenic V. cholerae in environmental water samples. The semi-nested PCR assay amplifi ed cholera toxin (ctxA2B) gene present in the toxigenic V. cholerae. The detection sensitivity of direct cell semi-nested PCR was 2 x 10 3 CFU of V. cholerae whereas direct cell single-step PCR could detect 2 x 10 4 CFU of V. cholerae. The performance of the assay was evaluated using environmental water samples after spiking with known number of Vibrio cholerae O1. The spiked water samples were fi ltered through a 0.22 micrometer membrane and the bacteria retained on fi lters were enriched in alkaline peptone water and then used directly in the PCR assay. The semi-nested PCR procedure coupled with enrichment could detect less than 1 CFU/ml in ground water and sea water whereas 2 CFU/ml and 20 CFU/ml could be detected in pond water and tap water, respectively. The proposed method is simple, faster than the conventional detection assays and can be used for screening of drinking water or environmental water samples for the presence of toxigenic V. cholerae.

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Detection of toxigenicVibrio choleraefrom environmental water samples by an enrichment broth cultivation-pit-stop semi-nested PCR procedure

Cited by 48 publications

References 44 publications

Molecular-Beacon Multiplex Real-Time PCR Assay for Detection of Vibrio cholerae

Molecular-Beacon Multiplex Real-Time PCR Assay for Detection of Vibrio cholerae

TaqMan PCR for Detection of Vibrio cholerae O1, O139, Non-O1, and Non-O139 in Pure Cultures, Raw Oysters, and Synthetic Seawater

Semi-nested polymerase chain reaction for detection of toxigenic Vibrio cholerae from environmental water samples

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