Analysis of lolB gene sequence and its use in the development of a PCR assay for the detection of Vibrio cholerae

Lalitha, P.; Suraiya, M.N. Siti; Lim, Keah-Ying; Lee, S.Y.; Haslindawaty, Abd Rashid Nur; Chan, Yean Yean; Ismail, Asma; Zafarina, Zainuddin; Ravichandran, M.

doi:10.1016/j.mimet.2008.05.001

Cited by 38 publications

(17 citation statements)

References 14 publications

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“…The hemM (lolB) gene was used to confirm the biochemical identification of V. cholerae (Fig. 1), since it is known to be highly conserved among all serogroups and biotypes of V. cholerae (20). The multiplex PCR also facilitated the analysis of the differences in DNA sequence of the tcpA gene, which was used to biotype the isolates.…”

Section: Resultsmentioning

confidence: 99%

Molecular Evidence of Cholera Outbreak Caused by a Toxigenic Vibrio cholerae O1 El Tor Variant Strain in Kelantan, Malaysia

Ang

Balqis

et al. 2010

J Clin Microbiol

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A total of 20 Vibrio cholerae isolates were recovered for investigation from a cholera outbreak in Kelantan, Malaysia, that occurred between November and December 2009. All isolates were biochemically characterized as V. cholerae serogroup O1 Ogawa of the El Tor biotype. They were found to be resistant to multiple antibiotics, including tetracycline, erythromycin, sulfamethoxazole-trimethoprim, streptomycin, penicillin G, and polymyxin B, with 35% of the isolates being resistant to ampicillin. All isolates were sensitive to ciprofloxacin, norfloxacin, chloramphenicol, gentamicin, and kanamycin. Multiplex PCR analysis confirmed the biochemical identification and revealed the presence of virulence genes, viz., ace, zot, and ctxA, in all of the isolates. Interestingly, the sequencing of the ctxB gene showed that the outbreak strain harbored the classical cholera toxin gene and therefore belongs to the newly assigned El Tor variant biotype. Clonal analysis by pulsed-field gel electrophoresis demonstrated that a single clone of a V. cholerae strain was responsible for this outbreak. Thus, we present the first molecular evidence that the toxigenic V. cholerae O1 El Tor variant has invaded Malaysia, highlighting the need for continuous monitoring to facilitate early interventions against any potential epidemic by this biotype.

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Section: Resultsmentioning

confidence: 99%

Molecular Evidence of Cholera Outbreak Caused by a Toxigenic Vibrio cholerae O1 El Tor Variant Strain in Kelantan, Malaysia

Ang

Balqis

et al. 2010

J Clin Microbiol

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“…Many virulence genes, such as omp , ctx , zot , ace , tcp , rtx , sto and hly in V. cholerae ; tdh , trh and toxR in V. parahaemolyticus ; and vvh , viuB and toxR in V. vulnificus, have been targeted for species‐specific detection by uniplex or multiplex PCR (Lalitha et al . ; Neogi et al . ; Teh et al .…”

Section: Introductionmentioning

confidence: 99%

“…Multiplex PCR has been proven to provide rapid and highly sensitive methods for the specific detection of micro-organisms (Fan et al 2008) and can be easily performed in diagnostic laboratories. Many virulence genes, such as omp, ctx, zot, ace, tcp, rtx, sto and hly in V. cholerae; tdh, trh and toxR in V. parahaemolyticus; and vvh, viuB and toxR in V. vulnificus, have been targeted for species-specific detection by uniplex or multiplex PCR (Lalitha et al 2008;Neogi et al 2010;Teh et al 2010). According to Neogi et al (2010), to accurately detect particular individual species, it is critical to address unresolved complications such as the precise differentiation of V. parahaemolyticus from closely related species, the simultaneous detection of all target species in a sample and the coexistence of V. cholerae, V. parahaemolyticus and V. vulnificus in costal environments, diseased animals, seafood or aquaculture (Gopal et al 2005;Mahmud et al 2008).…”

Section: Introductionmentioning

confidence: 99%

Development of agroELgene-based species-specific multiplex polymerase chain reaction assay for simultaneous detection ofVibrio cholerae,Vibrio parahaemolyticusandVibrio vulnificus

Hossain

Kim

et al. 2012

J Appl Microbiol

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Aims: To develop an effective multiplex polymerase chain reaction (PCR) for the simultaneous detection of three important Vibrio species, Vibrio cholerae (Vc), V. parahaemolyticus (Vp) and V. vulnificus (Vv) using the groEL gene, a potential phylogenetic marker. Methods and Results: Three species-specific primer sets were designed to target Vc, Vp and Vv. A total of 131 Vibrio and non-Vibrio strains were used to determine the specificity and sensitivity of primers. The primers produced specific PCR fragments from all target species strains and did not cross-react with other Vibrio and non-Vibrio species. This PCR method showed good efficiency in detecting coexisting target species in the same sample with a detection limit of 100 pg of Vc, Vp and Vv from mixed purified DNA. Detection of three target species was also possible from artificially inoculated shellfish, flounder and sea water. Conclusions:The groEL gene is a potential marker for accurate simultaneous detection of Vc, Vp and Vv and could be used to detect these species in environmental and clinical samples. Significance and Impact of the Study: This newly developed multiplex PCR is a useful and cost-effective method that is applicable in a disease-outbreak prediction system and may provide an effective tool for both the epidemiologist and ecologist.

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“…The most widely used genes for the detection of this species are toxR (the transcriptional regulator for the genes encoding the major outer membrane porins OmpU and OmpT) and ompW (outermembrane protein) (Baron, Chevalier, & Lesne, 2007;Goel, Tamrakar, Nema, Kamboj, & Singh, 2005;Gubala & Proll, 2006;Nandi et al, 2000;Neogi et al, 2010;Sheikh, Goodarzi, & Aslani, 2012;Shuan Ju Teh et al, 2009), even though recently the lolB (outermembrane lipoprotein) gene has also been described as an optimal target for V. cholerae detection (Chua et al, 2011;Lalitha et al, 2008). Out of these, the most extensively applied and evaluated gene was the ompW.…”

Section: Vibrio Choleraementioning

confidence: 99%

Detection of foodborne pathogens by qPCR: A practical approach for food industry applications

Chapela

Garrido‐Maestu

2015

Cogent Food & Agriculture

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Microbiological analysis of food is an integrated part of microbial safety management in the food chain. Monitoring and controlling foodborne pathogens are traditionally carried out by conventional microbiological methods based on culture-dependent approaches in control laboratories and private companies. However, polymerase chain reaction (PCR) has revolutionized microbiological analysis allowing detection of pathogenic microorganisms in food, without the necessity of classical isolation and identification. However, at present, PCR and quantitative polymerase chain reaction (qPCR) are essential analytical tools for researchers working in the field of foodborne pathogens. This manuscript reviews recently described qPCR methods applied for foodborne bacteria detection, serving as economical, safe, and reliable alternatives for application in the food industry and control laboratories. Multiplex qPCR, which allows the simultaneous detection of more than one pathogen in one single reaction, saving considerable effort, time, and money, is emphasized in the article.

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Analysis of lolB gene sequence and its use in the development of a PCR assay for the detection of Vibrio cholerae

Cited by 38 publications

References 14 publications

Molecular Evidence of Cholera Outbreak Caused by a Toxigenic Vibrio cholerae O1 El Tor Variant Strain in Kelantan, Malaysia

Molecular Evidence of Cholera Outbreak Caused by a Toxigenic Vibrio cholerae O1 El Tor Variant Strain in Kelantan, Malaysia

Development of agroELgene-based species-specific multiplex polymerase chain reaction assay for simultaneous detection ofVibrio cholerae,Vibrio parahaemolyticusandVibrio vulnificus

Detection of foodborne pathogens by qPCR: A practical approach for food industry applications

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