Study On The Rapid Detection Of Vibrio Parahaemolyticus In Seafood By Loop-mediated Isothermal Amplification (LAMP)

Background:Vibrio parahaemolyticus is a Gram-negative bacteria which is widely distributed in marine products and aquatic environment.It can cause astrointestinal illness,and even lead to fatal sapraemia.It is explicitly stipulated in both domestic and international regulations that the Vibrio parahaemolyticus cannot be detected in a lot of food.The current applied detection method is "GB 4789.7-2013 Microbiological examination of food hygiene--Examination of vibrio parahaemolyticus ".The procedures of the examination include pre-enrichment,selective enrichment,selective plate separation,biochemical identification and serology identification.It usually takes 5 to 7 days to finish the test,which is very time-consuming.The real-time fluorescence PCR can be used for detection of Vibrio parahaemolyticus,such as "SN/T 2424-2010 rapid detection and identification of Vibrio parahaemolyticus in import and export foods-real-time fluorescence PCR method" its procedures include enrichment,nucleic acid extraction and fluorescence PCR test and this method is rather simple and time-saving.However,due to the expensive equipment and professional technicians,it is difficult to meet the market’s demand.Loop-mediated isothermal amplification(LAMP)is a new isothermal nucleic acid amplification technology,developed by the Japanese Notomi etc.The technique is simple,fast,and has strong specificity.It allows one-step detection of target gene amplification at a single temperature after primers specifically recognize the target sequence.The amplification efficiency can be greatly increased after the introduction of the loop primers.Purpose:1.Based on V.parahaemolyticus-thermolabile hemolysin(thermolabile hemolysin,TLH)tlh gene,the primers were designed and screened out,the reaction system was also optimized to establishe a sensitive,specific,reproducible LAMP method for detecting V.parahaemolyticus.2.According to the optimized LAMP method,we established a rapid,sensitive,accurate,and simple LAMP detection system which may present a useful tool for detecting V.parahaemolyticus in seafood.Method:1.Based on Vibrio parahaemolyticus-thermolabile hemolysin(thermolabile hemolysin,TLH)tlh gene.In this paper,two sets of primers are designed and the set of primers which has shorter peak time,strong detection signal and without nonspecific amplification is also screened out.The specific gene primers specifically recognize the target sequence,start cycling strand displacement reaction under the action of Bst polymerase after adding the SYTO-9 fluorescent dye in the reaction system,which was incubated under the constant 60~65 ℃ temperature condition for 30 to 60 min.Then the target-specific sequence amplification shall be completed,and the reactions can also be monitored in real time.The established LAMP method was optimized by Mg2+ concentration,betaine concentration,d NTPs concentration and temperature.It was evaluated for its specificity,sensitivity and reproducibility.2.The system included optimized LAMP method,the developed Vibrio parahaemolyticus nucleic acid detection kit and Deaou-308 ℃ constant fluorescence detector.The LAMP system’s specificity and sensitivity was detected and evaluated for 1 standard strain of Vibrio parahaemolyticus,50 wild strains of Vibrio parahaemolyticus,8 near-source strains of Vibrio parahaemolyticus and 40 spiked seafood samples.Results:1.The established LAMP reaction mixture containing a 1.6μmol/L concentration of each inner primer(FIP,BIP),0.2μmol/L concentration of each ouer primer(F3,B3),0.8μmol/L concentration of each loop primer(LF,LB),6mmol/L concentration of Mg2+,8U of the Bst DNA polymerase large fragment,1.6 mmol/L each deoxynucleoside triphosphate,0.2μmol/L of SYTO-9,and 200 ng of DNA template;reaction temperature: 63 ℃;reaction time: 45 min.2.The assay correctly identified Vibrio parahaemolyticus,but did not detect 6 near-source strains of Vibrio parahaemolyticus and 14 strains of pathogenic bacteria.The result indicates the method has the advantage of specificity.3.The sensitivity for direct detection of Vibrio parahaemolyticus in pure cultures was 103 CFU / m L,and the sensitivity for plasmid DNA of Vibrio parahaemolyticus was up to 1fg / μl.4.Reproducible experimental results showed that test results were positive after 20 repeated experiments on the concentration above the detection limit of plasmid DNA of Vibrio parahaemolyticus,while the test results were negative after 20 times of repeated experiments on negative samples,showed good reproducibility.5.The LAMP detection system(includeing optimized LAMP method,Vibrio parahaemolyticus nucleic acid detection kit developed according to this method,and Deaou-308 ℃ constant fluorescence detector)successfully detected 1 standard strain of Vibrio parahaemolyticus and 50 wild strains of Vibrio parahaemolyticus,while showing negative results for 8 near-source strains of Vibrio parahaemolyticus,indicating that the detection system was highly specific.6.The detection sensitivity of the LAMP detection system for 40 spiked seafood samples(including processed seafood,fish and shellfish)was 103 CFU / m L.Conclusions:1.The developed LAMP rapid detection method targeting the tlh gene has the advantage of simplicity,rapidity,specificity and reproducity,which provides a new development direction for the detecting of V.parahaemolyticus.2.This rapid,accurate,and simple LAMP detection system(includeing optimized LAMP method,Vibrio parahaemolyticus nucleic acid detection kit and Deaou-308 ℃ constant fluorescence detector)may present a useful tool for detecting V.parahaemolyticus in seafood.