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.
Highlights
Vibrio parahaemolyticus and Vibrio vulnificus, which are the halophilic bacteria commonly found in the estuarine, coastal, and marine environments, belong to the family Vibrionaceae and cause seafood-borne gastrointestinal disorders in humans [1]
V. parahaemolyticus strains or V. vulnificus strains was processed in the absence or presence of genomic
The amplification products were analyzed by visual inspection using Fluorescent Detection Reagent (FD) reagent, V. parahaemolyticus strains or V. vulnificus strains was processed in the absence or presence of genomic and the positive in V. parahaemolyticusand V. by vulnificus-loop-mediated isothermal amplification (LAMP)
Summary
Vibrio parahaemolyticus and Vibrio vulnificus, which are the halophilic bacteria commonly found in the estuarine, coastal, and marine environments, belong to the family Vibrionaceae and cause seafood-borne gastrointestinal disorders in humans [1]. The LAMP technique have been adopted for detecting V. parahaemolyticus and V. vulnificus in seafood and environment samples [14,15] This amplification technology has been restricted to detect a single target, limiting the applicability of this assay [16,17]. We have devised multiple endonuclease restriction real-time loop-mediated isothermal amplification technique (MERT-LAMP), which overcame the limitations posed by current. We established a rapid, sensitive, and specific testing method based on the MERT-LAMP approach for simultaneous detection of V. parahaemolyticus and V. vulnificus by targeting toxR (GenBankID: L11929) and rpoS (GenBankID: AY187681) genes, respectively, and determined the sensitivity and specificity using pure cultures and spiked oyster samples
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
