Study On Multiplex Loop-mediated Isothermal Amplification Assay For The Detection Of Three Food-borne Pathogens

For the few past years,food safety problems emerge in endlessly which we get used to gradually.And at one time the people turn pale at the mention of food.Food-borne diseases are within the scope of our country or even the world's first food safety issues,and one of the major public health problems,causing economic burdens and social impact in countries around the world that difficult to measure.Among all the factors for food-borne diseases including microorganisms,chemical or physical agents,poisonous plants and animals,microbes are the most important factors that hold the first place.So food-borne pathogens which make up the majority of microorganisms are the most important problem with which China's public health and safety faced.Salmonella,the serotype of which are numerous,is the most common and second most important pathogenic bacteria in our country.Vibrio parahaemolyticus,found massively in coastal waters around the world,has became the first food-borne pathogens in China or even many Asian countries.Humans may become infected by ingesting of raw or cooked seafood.Listeria monocytogenes,which can cause fatal listeriosis,are common in Europe and the United States.And they were frequently found in recent years and have became one of the most common food-borne pathogens in China.For these three species of bacteria,it is necessary to establish a method for the rapid screening and diagnosis of them.In general,methods for rapid detection of food-borne pathogens include immunology methods,bio-sensor technology and nucleic acid-based detection.Immunology methods such as ELISE,IMS depend on the specific combination of antigen-antibody,and their anti-interference ability is poor.The shortcomings of bio-sensor technology are: low stability and high cost.PCR and PCR-based technologies,the classic representative of nucleic acid-based detection,have made great improvements in detection sensitivity and stability.But these methods are having more and more difficulty meeting real needs of instant diagnosis of infectious diseases owing to the dependence on the professional operation and precision apparatus such as thermal cycler.The emergence of isothermal nucleic acidamplification methods makes up the gap which need no temperature cycle,and nucleic acid-based detection from the lab to the field becomes possible.Loop-mediated isothermal amplification(LAMP),master of isothermal nucleic acid amplification methods,have a high specificity and a sensitivity equivalent to nest-PCR.The LAMP reaction involves an enzyme,and target sequences can be amplified within 60 min at a constant temperature.Rapid and continuous LAMP reaction leads to the high amplification efficiency.RNA can also be amplified through LAMP reaction.The LAMP products can be seen visually,and real-time monitoring the reaction can be performed with turbidity or fluorescence.Owing to the above advantages,the assay was widely applied since it was invented in various fields,such as pathogen identification,gene diagnosis and rapid detection of environmental samples.The study of the LAMP assay seems to have been very perfect except that once reaction can only detect a single target sequence at present.The development of multiplex LAMP approaches are limited.The reason for this can be attributed to two aspects of one “easy” and one “hard”.First,non-specific amplification can easily occur in multiplex LAMP reaction system;and second,multiple specific genes are hard to distinguish from multiplex LAMP reaction system.Due to the different size of the LAMP products,multiple sequences could not be differentiated from multiplex reaction system by agarose gel electrophoresis like multiplex PCR.These two factors is the bottleneck restricting the development of multiplex LAMP.In view of the above analysis,This study mainly has the following three parts:1.Development of loop-mediated isothermal amplification(LAMP)for detection of Salmonella,Vibrio parahaemolyticus,Listeria monocytogenesLAMP primers were designed to target the Vibrio parahaemolyticus tlh gene,the Salmonella bcfD gene and the Listeria monocytogenes iap gene,respectively.primers specificity were further validated by Blast.Taking into consideration of amplification efficiency and detection sensitivity,the optimal primers were selected and the LAMP reaction system was thus established.To test the detection of specificity,DNAs extracted from other similar food-borne pathogenic bacteria were used.And PCR was performed to determine and evaluate the detection sensitivity of LAMP.The real-time turbidity-based results showed that the amplification completed within 40 min.Specific test showed that three sets of primers of these three pathogens can only amplify the corresponding target sequences,indicating a high detection specificity.And a high sensitivity,or a sensitivity superior to PCR's sensitivity was confirmed in the comparison with that of PCR.The detection performance of the assay needs further investigated through application of the method to simulated samples or actual samples.2.Development of a multiplex loop-mediated isothermal amplification method for detection of Salmonella,Vibrio parahaemolyticus and Listeria monocytogenesThe aim of the study is to establish a multiplex LAMP reaction system for simultaneously detection of Salmonella,Vibrio parahaemolyticus and Listeria monocytogenes.Concept and consciousness of primer concentrations optimization was introduced.The optimized proportion of primer concentrations was determined by investigating the effects of three kinds of combinations of different primer concentrations on the amplification efficiency and by considering the cost.The multiplex LAMP reaction system was thus established,and its specificity and sensitivity were preliminary evaluated.The results showed that a multiplex LAMP reaction system combining 18 primers with concentrations of 1.3?mol/L can ensure the amplification efficiency and reduce the possibility of the occurence nonspecific amplification.The experimental results of specificity and sensitivity showed that the assay can specifically detect Salmonella strains or Vibrio parahaemolyticus strains or Listeria monocytogenes strains,and their mixture of genomic DNAs.The sensitivity of the multiplex LAMP was consistent with that of LAMP amplification only for Salmonella or Vibrio parahaemolyticus or Listeria monocytogenes,respectively.The multiplex LAMP to some extent reduce the workload and save the cost,and can be used as a primary screening method on a large scale.3.Development of a multiplex real-time LAMP assay for detection of Salmonella and Vibrio parahaemolyticusThis study is intended to develop a multiplex real-time LAMP assay for simultaneously detection of Salmonella and Vibrio parahaemolyticus so as to break the bottleneck of multiple specific genes are hard to distinguish from multiplex LAMP reaction system.Fluorescent dye EveGreen was added to the multiplex LAMP reaction system,and multiplex LAMP products were subject to melting curve analysis.Based on different melting temperatures(Tm values)of target sequences,Salmonella and Vibrio parahaemolyticus could be detected and differentiated simultaneously.The assay's sensitivity and specificity were preliminary assessment,and its quantitative ability was also studied.The experimental results showed that Tm values of amplified products are relatively stable of the Salmonella bcfD gene or the Vibrio parahaemolyticus toxR gene,which mean Tm values are 86.25 ± 0.06? and 84.05 ± 0.06?,respectively.Thus,the two target sequences could be distinguished by their different Tm values,which generated two peaks showed up in the melting curve.Specificity results of experimental demonstrated that the established assay could amplified all of Salmonella DNA or Vibrio parahaemolyticus DNA.For 7 Salmonella strains,their Tm values consistently fell between 86.5 and 86.8oC,suggesting that the real-time LAMP assay was highly specific.The sensitivity of the assay for simultaneously detection and differentiation Salmonella and Vibrio parahaemolyticus is consistent with that of multiplex PCR.The quantitative capability tests showed a linear correlation(R2=0.934)between the DNA concentrations and the Cq values,indicating the basic quantitative ability of the assay.The phenomenon of preferential amplification in the multiplex LAMP system was also observed and validated,and it was balanced by optimizing of primers concentrations on the basis of the principle of reduce the strong and increase the weak.In conclusion,This study aimed at three important food-borne pathogens of our country and turned to the research of LAMP technique,focusing on the destruction of two bottlenecks restricting the development of multiplex LAMP.So as to further optimize and improve the LAMP assay.Thus,technical support for rapid detection of food-borne pathogens is provided,and theoretical base for the multiple biological detection method is laid.