Development Of A Detection Method For Pathogens Based On Amino Modified Silica Coated Magnetic Nanoparticles And PCR

Foodborne pathogen is one of important factors which cause food-related human illness,and even lead to death.Currently,the main method for pathogen detection is culture method,the total process normally lasts 5-7 days.Owing that it is time-consuming,it is limited sometimes in practical applications.Thus,the development of rapid detection methods for foodborne pathogens has an important significance on food safety.In this study,molecular methods were selected to detect foodborne pathogens.As we all known,genomic DNA is the key material for organism.With the rapid development of genomics and computer science,molecular detection will play more and more important role.However,when molecular methods were used in the field of food safety,the reactions were always inhibited by food matrix.In present study,we used magnetic nanoparticles to separate the target genetic DNA from food samples,then the molecular methods were used to detect foodborne pathogens.That is,combining with magnetic separation,the molecular methods will be able to exert their full advantages on food safety.The main contents and results are as follows.1.We prepared several types of magnetic nanoparticles,and characterized them by TEM,IR,and so on.We used three types of nanoparticles to adsorb the DNA from 1×PCR buffer.The result showed that ASMNP have remarkable advantage to separate DNA(both dsDNA and ssDNA).After exploring the effects of temperature and pH,the results indicated that electrostatic interactions and hydrogen bonds might played significant roles.2.We explored the effect of ASMNP on PCR about inhibition,specificity,efficiency and yield.We found that:1)Inhibition:nanoparticles inhibited PCR by adsorbing PCR components.When the surface was blocked,the inhibition would be eliminated.2)Specificity:Nanoparticles were not able to inhibit the nonspecific products which caused by false priming.However,ASMNP inhibited the nonspecific products caused by premature termination.3)Efficiency and Yield:some nanoparticles which adsorbed ssDNA more strongly than dsDNA could facilitate the melting of dsDNA and prevent the ssDNA from renaturation.For PCR,the original available ssDNA was very important for the resulting yield.Overall,the effect of nanoparticles on PCR was the result caused by multiple factors.For our purpose,the effect of nanoparticles on PCR was mainly achieved through the surface of nanoparticles.If nanoparticles was blocked effectively,the complex would not affect the PCR.3.In this part,a method based on ASMNP and polymerase chain reaction(PCR)was developed to rapidly and sensitively detect foodborne pathogens in raw milk.A trace amount of genomic DNA of pathogens was extracted directly from complex matrices such as raw milk using ASMNP.The magnetically separated complexes of genomic DNA and ASMNP were directly subjected to single PCR(S-PCR)or multiplex PCR(M-PCR)to detect single or multiple pathogens from raw milk samples.Salmonella Enteritidis(Gram-negative)and Listeria monocytogenes(Gram-positive)were used as model organisms to artificially contaminate raw milk samples.After magnetic separation and S-PCR,the detection sensitivities were 8CFU/mL and 13 CFU/mL respectively.Furthermore,this method was successfully used to detect multiple pathogens(S.Enteritidis and L.monocytogenes)from artificially contaminated raw milk using M-PCR at sensitivities of 15 CFU/mL and 25 CFU/mL,respectively.4.Functionalization of magnetic nanoparticles is a key factor in allowing efficient capture of the target analytes.In this part,we reported the synthesis of a type of amino-rich silica coated magnetic nanoparticles using a one-pot method.This type of magnetic nanoparticle had a rough network surface and a higher density of amino groups than the nanoparticles prepared by the post-modification method.Furthermore,the results of hydrochloric acid treatment indicated that the magnetic nanoparticles were stably coated.The developed amino-rich silica coated magnetic nanoparticles were labeled with oligonucleotides to capture target sequences.The results of real-time quantitative PCR showed that the magnetic capture probes based on nanoparticles with higher amino group density resulted in better separation efficiency.5.In this part,ARSMNP were labeled with oligonucleotides for capturing mRNA.And the details were optimized.We simply treated with the milk samples,then,separated the target mRNA using the magnetic probes,finally detected Salmonella in milk samples by RT-qPCR.The sensitivity was about 10~4 CFU/mL.In order to obtain lower detection limit,we enriched the target pathogens by culture methods for overnight.In this case,the sensitivity could reach 10 CFU/mL.