{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,28]],"date-time":"2026-08-28T04:28:38Z","timestamp":1787891318173,"version":"build-2784847793"},"reference-count":22,"publisher":"Microbiology Society","issue":"4","content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2011,4,1]]},"abstract":"<jats:p><jats:italic>Vibrio cholerae<\/jats:italic>has caused severe outbreaks of cholera worldwide with thousands of recorded deaths annually. Molecular diagnosis for cholera has become increasingly important for rapid detection of cholera as the conventional methods are time-consuming and labour intensive. However, traditional PCR tests still require cold-chain transportation and storage as well as trained personnel to perform, which makes them user-unfriendly. The aim of this study was to develop a thermostabilized triplex PCR test for cholera which is in a ready-to-use form and requires no cold chain. The PCR test specifically detects both toxigenic and non-toxigenic strains of<jats:italic>V. cholerae<\/jats:italic>based on the cholera toxin A (<jats:italic>ctxA<\/jats:italic>) and outer-membrane lipoprotein (<jats:italic>lolB<\/jats:italic>) genes. The thermostabilized triplex PCR also incorporates an internal amplification control that helps to check for PCR inhibitors in samples. PCR reagents and the specific primers were lyophilized into a pellet form in the presence of trehalose, which acts as an enzyme stabilizer. The triplex PCR was validated with 174 bacteria-spiked stool specimens and was found to be 100\u200a% sensitive and specific. The stability of the thermostabilized PCR was evaluated using the Q<jats:sub>10<\/jats:sub>method and it was found to be stable for approximately 7 months at 24\u2005\u00b0C. The limit of detection of the thermostabilized triplex PCR assay was 2\u00d710<jats:sup>4<\/jats:sup>c.f.u. at the bacterial cell level and 100\u2005pg DNA at the genomic DNA level, comparable to conventional PCR methods. In conclusion, a rapid thermostabilized triplex PCR assay was developed for detecting toxigenic and non-toxigenic<jats:italic>V. cholerae<\/jats:italic>which requires minimal pipetting steps and is cold chain-free.<\/jats:p>","DOI":"10.1099\/jmm.0.027433-0","type":"journal-article","created":{"date-parts":[[2010,12,24]],"date-time":"2010-12-24T03:21:36Z","timestamp":1293160896000},"page":"481-485","source":"Crossref","is-referenced-by-count":41,"title":["Development of a dry reagent-based triplex PCR for the detection of toxigenic and non-toxigenic Vibrio cholerae"],"prefix":"10.1099","volume":"60","author":[{"given":"Ang Lim","family":"Chua","sequence":"first","affiliation":[{"name":"School of Health Sciences, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Husni Tan","family":"Elina","sequence":"additional","affiliation":[{"name":"School of Medical Sciences, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Boon Huat","family":"Lim","sequence":"additional","affiliation":[{"name":"School of Health Sciences, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chan Yean","family":"Yean","sequence":"additional","affiliation":[{"name":"School of Medical Sciences, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Manickam","family":"Ravichandran","sequence":"additional","affiliation":[{"name":"Department of Biotechnology, Faculty of Applied Sciences, AIMST University, Semeling, 08100 Bedong, Kedah, Malaysia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Pattabhiraman","family":"Lalitha","sequence":"additional","affiliation":[{"name":"Department of Biotechnology, Faculty of Applied Sciences, AIMST University, Semeling, 08100 Bedong, Kedah, Malaysia"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"345","reference":[{"key":"R1","doi-asserted-by":"crossref","first-page":"1633","DOI":"10.1128\/JCM.35.6.1633-1635.1997","article-title":"Rapid detection of Vibrio cholerae O139 Bengal from stool specimens by PCR","volume":"35","author":"Albert","year":"1997","journal-title":"J Clin Microbiol"},{"key":"R2","doi-asserted-by":"crossref","first-page":"373","DOI":"10.1016\/j.diagmicrobio.2007.05.014","article-title":"Amplification of ST50 gene using dry-reagent-based polymerase chain reaction for the detection of Salmonella typhi","volume":"59","author":"Aziah","year":"2007","journal-title":"Diagn Microbiol Infect Dis"},{"key":"R3","volume-title":"Laboratory Methods for the Diagnosis of Epidemic Dysentery and Cholera . 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