Study On A Loop Mediated Isothermal Amplification (LAMP) For The Rapid Detection Of Pathogens In Aquatic Animals

With the scale expansion of aquaculture and degradation of aquatic environment, diseases of aquatic animals has become more and more serious, which is one of the bottlenecks of the aquaculture development. Rapid diagnosis of aquatic pathogens was of great significance for breeding, disease prevention, import and export quarantine, aquatic food safety, etc. Since their individual deficiency, most of the conventional pathogen detection techniques are not suitable for rapid field detection. Loop-mediated isothermal amplification (LAMP) is a new DNA amplification method. It relies on the automatic cycle of the strand displacement reaction. With a set of primers targeting six specific regions and DNA polymerase with strand displacement, it can amplify target nucleic acids from a few copies to109copies within1h. High specificity, sensitivity and efficiency, with easy protocol, make LAMP technique suitable for rapid field detection.In this study, LAMP methods for rapid detection of white spot syndrome virus (WSSV), infectious hypodermal and haematopoietic necrosis virus (IHHNV) and Vibrios were developed. Multiplex LAMP for detecting WSSV and IHHNV at the same time and universal LAMP for detecting a group of specific pathogens were developed for the first time. The universal LAMP was used to detect Vibrio spp. Different primers were designed to targeting different pathogens DNA sequences. LAMP reaction conditions were optimized, including concentration of magnesium ions, the betaine concentration, reaction time, reaction temperature, etc. And the specificity and sensitivity were tested. DUTP-UNG (uracil-N-glycosylase) system was used to overcome carry-over contamination of LAMP for the first time. The replacement rate of dUTP to dTTP and the influence of the replacement to LAMP sensitivity were studied. LAMP detection systems were used for aquatic animal samples detection. Specific conclusions are as follows:1. Incubating the optimized LAMP reaction for WSSV at64℃for45min can have the effect amplification. By adding SYBR Green I, the results can be judged by naked eyes. Using common bacteria and shrimp viruses as controls, LAMP method has good specificity WSSV detection. Sensitivity of LAMP was100copies/μl, which was100times higher than the nested PCR. DUTP replacing part of dTTP in the reaction mixture, combining the UNG enzyme, LAMP carry-over contamination can be prevented. But when the replacement rate was higher than50%, amplification efficiency was significantly inhibited. The LAMP method has higher sensitivity and faster detection process than nested PCR in shrimp samples detection against WSSV.2. LAMP reaction system for IHHNV amplified properly at65℃for60min. Reaction tube color changed after staining, that makes it convenience for judgment. Expected fragments appeared after amplicons were digested by restriction enzyme. Fifty percent of the dUTP substitution and the using of the UNG can avoid the false positives, but that made the sensitivity decrease by about10times. LAMP detection showed good specificity for IHHNV. Its sensitivity was1000times higher than common PCR. In shrimp samples IHHNV detection, the LAMP method was consistent with PCR, but showed a higher efficiency and more intuitive results.3. Optimized mLAMP system for WSSV and IHHNV detection was incubated at65℃for60min to complete the reaction. After digestion, fragments of amplicons were consistent with the expectation. Multiplex LAMP has high specificity and sensitivity, which was100times higher than nested PCR and1000times higher than the conventional PCR, respectively. Multiplex LAMP detection showed higher detection rates and higher efficiency than the PCR assay in the detection of the shrimp sample.4. Optimized Vibrio spp. universal LAMP detection system need incubated at62℃for60min. Universal LAMP with a highly specificity for Vibrio spp. detection. The detection sensitivity was100times higher than the PCR method. The universal LAMP method showed very good detection ability when used in the detection of Vibrio from artificial infected aquatic animals.The results showed that the LAMP method is a simple, sensitivity and high specificity for the rapid pathogen detection. With no need for special equipment, it suitable for rapid detection kit development. LAMP method was a significant potential tool for high throughout and fast diagnosis against aquatic pathogens in the field.