Detection of Viable Toxigenic Vibrio cholerae from Environmental Water Sources by Direct Cell Duplex PCR Assay

Goel, Ajay; Tamrakar, Akhilesh K.; Nema, Vijay; Kamboj, Dev Vrat; Singh, Lokendra

doi:10.1007/s11274-004-7317-4

Cited by 24 publications

(19 citation statements)

References 13 publications

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“…Enrichment of V. cholerae in APW for 6 h was sufficient to multiply the bacteria to within the limits of sensitivity of PCR. In earlier studies, it was found that about a single CFU/mℓ of V. cholerae could be detected in water after 6 h enrichment in APW (Goel et al, 2005b). All 14 samples tested positive on TCBS plates and using other biochemical tests, which indicated that environmental strains contained non-O1 V. cholerae.…”

Section: Resultsmentioning

confidence: 96%

Detection and confirmation of toxigenic <i>Vibrio cholerae</i> O1 in environmental and clinical samples by a direct cell multiplex PCR

Yadava¹,

Jain²,

Ak³

2013

WSA

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Epidemic cholera caused by toxigenic Vibrio cholerae O1 is a major health problem in several developing countries. Traditional methods for identifying V. cholerae involve cultural, biochemical and immunological assays which are cumbersome and often take several days to complete. In the present study, a direct cell multiplex PCR was developed targeting the ompW, ctxB and rfbO1 genes for confirmation of V. cholerae, its toxigenicity and serogroup O1, respectively from clinical and environmental samples. The detection sensitivity of the multiplex PCR was 1.9 x 10 3 V. cholerae per PCR reaction. A total of 31 environmental samples and 45 clinical V. cholerae isolates from different outbreaks were examined by the PCR. The assay was simple and specific, as there was no requirement for DNA extraction and no amplification was observed with other homologous bacteria used. The assay can be very useful for rapid surveillance of toxigenic V. cholerae O1 in environmental water samples, as well as for confirmation of clinical isolates.

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Section: Resultsmentioning

confidence: 96%

Detection and confirmation of toxigenic <i>Vibrio cholerae</i> O1 in environmental and clinical samples by a direct cell multiplex PCR

Yadava¹,

Jain²,

Ak³

2013

WSA

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“…2 Monitoring of the bacterium in water sources is traditionally performed by a series of biochemical tests. 3,4 In addition, several immunoassay procedures employed for the detection of V. cholerae are: polymerase chain reaction (PCR), [5][6][7][8] enzyme linked immunosorbent assay (ELISA), 9 coaglutination, 10 immunofluorescence [11][12][13] and quartz crystal microbalance (QCM). 14 Most of these methods somehow require highly qualified personnel (PCR), long time (ELISA) and sophisticated instrumentation (QCM, PCR).…”

Section: Introductionmentioning

confidence: 99%

Amperometric Immunosensor for the Detection of Vibrio cholerae O1 Using Disposable Screen-printed Electrodes

et al. 2006

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A disposable amperometric immunosensor was studied for the rapid detection of Vibrio cholerae (V. cholerae), the causative agent of cholera, employing an indirect sandwich enzyme linked immunosorbent assay (ELISA) principle. Screen-printed electrodes (SPEs) were fabricated (by using commercial and homemade carbon inks), electrochemically characterized and the assay conditions were optimized for capturing antibodies and antigen. Whole cell lysate (WCL) of V. cholerae was used to raise antibodies in rabbits and mice. The antibodies raised against WCL of V. cholerae were found to be specific, and no cross reactivity was observed with other enteric bacteria. 1-Naphthyl phosphate was used as a substrate with the amperometric detection of its enzymatic hydrolysis product 1-naphthol at a potential of +400 mV vs. Ag/AgCl reference electrode. A comparison between the amperometric detection technique and the standard ELISA was made in terms of the total assay time, the amount of biological materials used and the sensitivity of detection. The minimum detection limit of the amperometric immunosensor for V. cholerae was found to be 10 5 cells/ml in 55 min, while ELISA detected 10 6 cells/ml in 4 h.

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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%

“…These environmental water samples were found positive for V. cholerae on TCBS agar plates and by other biochemical tests (data not shown). Our earlier study has also shown that most of the environmental V. cholerae strains are non-cholera toxin producers 7,14 . In this study, sensitivity of PCR was enhanced at least 10 times than the normal PCR.…”

Section: Resultsmentioning

confidence: 77%

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 Viable Toxigenic Vibrio cholerae from Environmental Water Sources by Direct Cell Duplex PCR Assay

Cited by 24 publications

References 13 publications

Detection and confirmation of toxigenic <i>Vibrio cholerae</i> O1 in environmental and clinical samples by a direct cell multiplex PCR

Detection and confirmation of toxigenic <i>Vibrio cholerae</i> O1 in environmental and clinical samples by a direct cell multiplex PCR

Amperometric Immunosensor for the Detection of Vibrio cholerae O1 Using Disposable Screen-printed Electrodes

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

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