Molecular-Beacon Multiplex Real-Time PCR Assay for Detection of
            <i>Vibrio cholerae</i>

Gubala, Aneta J.; Proll, David

doi:10.1128/aem.02597-05

Cited by 62 publications

(33 citation statements)

References 27 publications

(21 reference statements)

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“…The occurrence of drug resistance genes in microbial isolates will result in microorganisms that are multi-resistant to virtually all antimicrobial agent. This poses a serious public health concern because should such a multi-drug resistant organism cause infection, empirical treatment will be difficult (57,58,62 (73) is higher than the level presented in this work. Manganese was detected in the sea snail sample at 6.36 ppm.…”

Section: Discussioncontrasting

confidence: 38%

Comparative study of microbial, proximate and heavy metal compositions of some gastropods, bivalve and crustacean seafood

Oranusi¹,

Effiong²,

Duru³

2018

Af J Clin Exp Micro

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Background: Seafood are known to be nutritionally rich, however, water bodies from where these foods are harvested are often burdened with wastes from industries, household and run-offs. Thus seafood can harbor pathogenic microorganisms and heavy metals which can pose a serious health hazard to consumers. Materials and Methods: In this study, 5kg each of Littorina littorea, Achatina fulica,Tympanotonos fuscatus, Dorsanum miran, Egeria radiate and Penaeus notialis were investigated for microbial, proximate and heavy metal qualities using standard microbiological and chemical methods of analysis. Results: The gastropod mollusks Tympanotonos fuscatus, Littorina littorea and Achatina fulica had significantly (p≤0.05) higher microbial loads compared to other samples. All the samples had significantly (p≤0.05) higher coliform counts compared to standard specifications. The microbial isolates from the samples included species of Bacillus, Klebsiellae, Citrobacter, Providencia, Pseudomonas, Staphylococcus aureus, Escherichia coli and Aspergillus niger, Aspergillus flavus, yeasts, Alternaria, Absidia, Rhizopus, and Mucor. Antibiogram of the isolates revealed multiple resistances with 79.55% and 66.67% resistance to antibacterial and antifungal agents respectively. The seafood was rich in protein (8.0±0.03 to 46.0±0.03%) and fat (0.6±0.01 to 8.8±1.00%) but low in carbohydrate (1.18±0.84 to 3.81±0.86%).The heavy metal compositions of the seafood were generally low except for manganese 6.36±0.03ppm in Dorsanum miran and nickel 0.81±0.50 ppm in Penaeus notialis. Conclusion: The microbial loads of the seafood are significantly (p≤0.05) higher in gastropods and more than acceptable standard, effective protection of water bodies for food cultivation and constant monitoring of seafood is necessary. Adequate processing and employing good manufacturing practices can reduce the microbial loads to an acceptable level and prevent food borne hazards that could be associated with seafood consumption. Keywords: Seafood; Heavy metals; Microbial profile; proximate compositions ÉTUDE COMPARATIVE DE LA PROLIFÉRATION MICROBIENNE, PROCHAINE ET HEAVY METAL COMPOSITIONS DE CERTAINS GASTÉROPODES, MOLLUSQUES ET CRUSTACÉS LES FRUITS DE MER

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Section: Discussioncontrasting

confidence: 38%

Comparative study of microbial, proximate and heavy metal compositions of some gastropods, bivalve and crustacean seafood

Oranusi¹,

Effiong²,

Duru³

2018

Af J Clin Exp Micro

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“…A report of the use of molecular beacons for the multiplex real-time PCR detection of V. cholerae cells has recently been published, stating a detection limit of 5 ϫ 10 3 CFU/ml (25). The sensitivity of our assay proved to be 100-fold higher (50 CFU/ml).…”

Section: Discussionmentioning

confidence: 91%

“…The tcpA marker used in PCR was designed to detect the toxigenic El Tor strains. However, in one of these strains no amplification was detected (25). To our knowledge, these two studies are the only studies describing the use of molecular beacon probes for the detection of V. cholerae.…”

Section: Discussionmentioning

confidence: 92%

“…A direct comparison of the specificity of NASBA and real-time PCR using molecular beacons is difficult since different targets and different strains of bacteria were used. A real-time PCR assay using molecular beacons used four different genetic targets in a multiplex assay (rtxA, epsM, ompW, and tcpA) in order to detect V. cholerae cells; in this assay, V. mimicus was detected nonspecifically through ompW (25). The tcpA marker used in PCR was designed to detect the toxigenic El Tor strains.…”

Section: Discussionmentioning

confidence: 99%

“…PCR has now become a frequently used detection method, and several PCR protocols have been developed for V. cholerae (24,25,35,36,48,49). PCR detection confirms the presence of specific genetic regions in a target organism.…”

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confidence: 99%

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

Fykse

Skogan

Davies

et al. 2007

Appl Environ Microbiol

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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.Vibrio cholerae is the etiological agent of epidemic cholera, which causes watery diarrhea that can result in the rapid dehydration and death of infected persons. Coastal waters are an important reservoir of V. cholerae, and cholera is generally transmitted to humans via contaminated water or seafood (12, 13). Of more than 200 known serogroups of V. cholerae, the two well-known serogroups O1 and O139 have been associated with epidemic cholera (11). The serogroup O1 can be divided into three serotypes, Inaba, Ogawa, and Hikojima, and each serotype can be divided into two biotypes, classical and El Tor (30). The other serogroups of V. cholerae, collectively referred to as non-O1 and non-O139 serogroups, have not been associated with epidemics but have been associated with occasional outbreaks of cholera-like disease. These other serogroups are usually isolated from patients with mild diarrhea or from the environment (27,39). Vibrio species such as V. parahaemolyticus, V. cholerae, and V. vulnificus are found in blue mussels harvested along the coastline of Norway (5).The pathogenesis of cholera is a complex process, and the major virulence factors of V. cholerae are the cholera toxin (CT) encoded by the ctxAB genes and the toxin-coregulated pilus (TCP) encoded by the tcpA gene (30). CT leads to increased intestinal secretion of electrolytes and water into the lumen. TCP is a ...

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Detection, Isolation, and Identification of Vibrio cholerae from the Environment

et al. 2012

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Recent molecular advances in microbiology have greatly improved the detection of bacterial pathogens in the environment. These improvements and a downward trend in the cost of molecular detection methods have contributed to increased frequency of detection of pathogenic microorganisms where traditional culture-based detection methods have failed. Culture methods also have been greatly improved, and the confluence of the two suites of methods provides a powerful tool for detection, isolation, and characterization of pathogens. While molecular detection provides data on the presence and type of pathogens, culturing methods allow a researcher to preserve the organism of interest for "-omics" studies, such as genomic, metabolomic, secretomic, and transcriptomic analysis, which are rapidly becoming more affordable. This has yielded a clearer understanding of the ecology and epidemiology of microorganisms that cause disease. In this unit, we present commonly accepted methods for isolation, detection, and characterization of V. cholerae, providing more extensive knowledge of the ecology and epidemiology of this organism. This unit has been fully revised and updated from the earlier version with the latest knowledge and additional information not previously included.

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

Cited by 62 publications

References 27 publications

Comparative study of microbial, proximate and heavy metal compositions of some gastropods, bivalve and crustacean seafood

Comparative study of microbial, proximate and heavy metal compositions of some gastropods, bivalve and crustacean seafood

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

Detection, Isolation, and Identification of Vibrio cholerae from the Environment

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