title: Rapid and Sensitive Detection of Vibrio parahaemolyticus and Vibrio vulnificus by Multiple Endonuclease Restriction Real-Time Loop-Mediated Isothermal Amplification Technique.
abstract: Vibrio parahaemolyticus and Vibrio vulnificus are two marine seafood-borne pathogens causing severe illnesses in humans and aquatic animals. In this study, a recently developed novel multiple endonuclease restriction real-time loop-mediated isothermal amplification technology (MERT-LAMP) were successfully developed and evaluated for simultaneous detection of V. parahaemolyticus and V. vulnificus strains in only a single reaction. Two MERT-LAMP primer sets were designed to specifically target toxR gene of V. parahaemolyticus and rpoS gene of V. vulnificus. The MERT-LAMP reactions were conducted at 62 °C, and the positive results were produced in as short as 19 min with the genomic DNA templates extracted from the V. parahaemolyticus and V. vulnificus strains. The two target pathogens present in the same sample could be simultaneously detected and correctly differentiated based on distinct fluorescence curves in a real-time format. The sensitivity of MERT-LAMP assay was 250 fg and 125 fg DNA per reaction with genomic templates of V. parahaemolyticus and V. vulnificus strains, which was in conformity with conventional LAMP detection. Compared with PCR-based techniques, the MERT-LAMP technology was 100- and 10-fold more sensitive than that of PCR and qPCR methods. Moreover, the limit of detection of MERT-LAMP approach for V. parahaemolyticus isolates and V. vulnificus isolates detection in artificially-contaminated oyster samples was 92 CFU and 83 CFU per reaction. In conclusion, the MERT-LAMP assay presented here was a rapid, specific, and sensitive tool for the detection of V. parahaemolyticus and V. vulnificus, and could be adopted for simultaneous screening of V. parahaemolyticus and V. vulnificus in a wide variety of samples.
authors.affiliation: State Key Laboratory of Infectious Disease Prevention and Control, National Institute for Communicable Disease Control and Prevention, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Chinese Center for Disease Control and Prevention, Changping, Changbai Road 155, Beijing 102206, China. wildwolf0101@163.com.
authors.forename: Yi
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Changping District Center for Disease Control and Prevention, Changping, Beijing 102200, China. xuner1989@163.com.
authors.forename: Dongxun
authors.initials: D
authors.lastname: Li
authors.affiliation: State Key Laboratory of Infectious Disease Prevention and Control, National Institute for Communicable Disease Control and Prevention, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Chinese Center for Disease Control and Prevention, Changping, Changbai Road 155, Beijing 102206, China. wangyan@icdc.cn.
authors.forename: Yan
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Institute of Microbiology, Jilin Provincial Center for Disease Control and Prevention, Changchun, Jilin 130062, China. likeweihaotingting@163.com.
authors.forename: Kewei
authors.initials: K
authors.lastname: Li
authors.affiliation: State Key Laboratory of Infectious Disease Prevention and Control, National Institute for Communicable Disease Control and Prevention, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Chinese Center for Disease Control and Prevention, Changping, Changbai Road 155, Beijing 102206, China. yechangyun@icdc.cn.
authors.forename: Changyun
authors.initials: C
authors.lastname: Ye
mesh_terms: D000818:Animals; D004262:DNA Restriction Enzymes / Q000235:genetics*; D005506:Food Contamination / Q000032:analysis; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D010125:Ostreidae / Q000382:microbiology; D016133:Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity; D014736:Vibrio parahaemolyticus / Q000235:genetics*; D041261:Vibrio vulnificus / Q000235:genetics*
chemical_list: D004262:DNA Restriction Enzymes
keywords: LAMP; LoD; MERT-LAMP; V. parahaemolyticus; V. vulnificus