title: Identification of Vibrio cholerae serotypes in high-risk marine products with non-gel sieving capillary electrophoresis.
abstract: Vibrio cholerae, a natural inhabitant of the marine environment, poses a threat to human health, and its new epidemic variants have been reported. A method of multiplex polymerase chain reaction-capillary electrophoresis-laser-induced fluorescence (PCR-CE-LIF) detection has been developed to detect and identify V. cholerae in marine products sensitively, rapidly, and reliably. Four sets of primers were selected to amplify genus-specific VCC gene, O139 serogroup-specific O139 gene, O1 serogroup-specific O1 gene, and ctxA gene associated with the CT toxin of enterotoxigenic V. cholerae. The PCR products were detected using CE-LIF with SYBR Gold serving as the DNA fluorescent dye. The parameters of PCR and the separation conditions of CE-LIF were optimized. Under the optimal conditions, V. cholerae was detected and four serotypes were identified simultaneously within 8 min. The alignment analysis showed that the PCR products had good agreement with the published sequences from GenBank, indicating that the primers selected in this study had high specificity and the PCR results were reliable. The proposed method could detect 5 to 20 cfu/ml V. cholerae. The intraday precisions of migration time and peak area of DNA marker and PCR products were in the ranges of 1.60-2.56% and 1.60-6.29%, respectively. The specificity results showed that only five standard bacteria used in this study showed the specific peaks when the target bacteria were mixed with seven other common intestinal pathogenic bacteria at the same concentration. The assay was applied to 71 high-risk marine products, and different serotypes of V. cholerae could be identified sensitively and reliably.
authors.affiliation: West China School of Public Health, Sichuan University, Chengdu, Sichuan 610041, China.
authors.forename: Chen
authors.initials: C
authors.lastname: Zhou
authors.affiliation: Chengdu Center for Disease Control and Prevention, Chengdu, Sichuan 610041, China.
authors.forename: Ming
authors.initials: M
authors.lastname: Li
authors.affiliation: West China School of Public Health, Sichuan University, Chengdu, Sichuan 610041, China.
authors.forename: Chengjun
authors.initials: C
authors.lastname: Sun
authors.affiliation: Chengdu Center for Disease Control and Prevention, Chengdu, Sichuan 610041, China.
authors.forename: Haimin
authors.initials: H
authors.lastname: Zou
authors.affiliation: Jiangxi Provincial Institute for Food and Drug Control, Nanchang 330029, China.
authors.forename: Xin
authors.initials: X
authors.lastname: Wu
authors.affiliation: West China School of Public Health, Sichuan University, Chengdu, Sichuan 610041, China.
authors.forename: Liyin
authors.initials: L
authors.lastname: Zhang
authors.affiliation: West China School of Public Health, Sichuan University, Chengdu, Sichuan 610041, China.
authors.forename: Siyuan
authors.initials: S
authors.lastname: Tao
authors.affiliation: West China School of Public Health, Sichuan University, Chengdu, Sichuan 610041, China.
authors.forename: Bingyue
authors.initials: B
authors.lastname: Wang
authors.affiliation: West China School of Public Health, Sichuan University, Chengdu, Sichuan 610041, China. Electronic address: lyxlee2008@hotmail.com.
authors.forename: Yongxin
authors.initials: Y
authors.lastname: Li
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics; D001431:Bacteriological Techniques / Q000379:methods*; D052160:Benzothiazoles; D002772:Cholera Toxin / Q000235:genetics; D004269:DNA, Bacterial / Q000032:analysis* / Q000737:chemistry; D003959:Diamines; D019075:Electrophoresis, Capillary; D005456:Fluorescent Dyes / Q000737:chemistry; D060885:Multiplex Polymerase Chain Reaction; D009930:Organic Chemicals / Q000737:chemistry; D011804:Quinolines; D065288:Serogroup; D012703:Serotyping / Q000295:instrumentation; D013050:Spectrometry, Fluorescence; D014734:Vibrio cholerae / Q000235:genetics*
chemical_list: D001426:Bacterial Proteins; D052160:Benzothiazoles; D004269:DNA, Bacterial; D003959:Diamines; D005456:Fluorescent Dyes; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I; D002772:Cholera Toxin
keywords: Laser-induced fluorescence detection; Marine products; Multiplex PCR; Non-gel sieving capillary electrophoresis; Vibrio cholerae