Reaserch On Quantifying And Silencing Of Antibiotic Resistance Genes In Food

The emergence and spread of antibiotic resistant foodborne bacterial is mainly caused by antibiotics used in animal husbandry,it has become a public health concern in food industry,as it will increase the difficulties of clinic treatments.Studies indicated that the antibiotic resistant bacterial could be potentially transmitted to human through environmental pollution,food products industry chain and so on,which may be a threat to human health.This study aimed to investigated pollution status of resistant genes in market aquatic products,vegetables,fruits,meat and swine feces,do the quantitative analysis of resistant genes using Quantitative Real-time PCR method under the total sample DNA level and using siRNA to inhibit the expression of integrase 1 gene int I1 to find a new way to solve the spread of antibiotic resistant genes caused by intI1.The results are as follows:(1)12 class ARGs has been detected in 48 aquatic products,43 vegetable samples and 16 fruit samples.For aquatic products,the abundance of key genes ranked from 1.86×102 copies/g(cmlA,fish sample,A29)to 8.98×108 copies/g(tet B,shrimp sample,A22);the abundance of key genes in vegetables ranked from 2.07×102 copies/g(sul?,watercress,V18)to2.17×107 copies/g(tetM,green vegetables,V29);for fruit samples,the abundance of key genes ranked from 1.92×102 copies/g(ermB,pear,F12)to 1.07×107 copies/g(tetM,orange,F13).Tetracycline resistant genes were found to have highest concentration among six kinds of resistant genes in three kinds of samples,which detailed number were: 2.11×109 copies/g ?6.33×107 copies/g and 5.92×107 copies/g.Among three kinds of sample place,vegetable market got the most serious pollution of antibiotic resistant genes,then came to the aquatic products market and supermarket had the lowest concentration.(2)12 class ARGs has been detected in 13 meat products and 10 swine feces samples.For meat products,the abundance of key genes ranked from 2.31×102 copies/g(ermB,pork meat sample,M5)to 7.25×108 copies/g(tetA,pork meat sample,M1);the abundance of key genes in swine feces samples ranked from 3.04×102 copies/g(ermB,swine feces sample,S5)to1.59×108 copies/g(aadA,swine feces sample,S4).Tetracycline resistant genes were found to have highest concentration among six kinds of resistant genes in three kinds of samples,which detailed number were: 8.26×108 copies/g and 5.96×108 copies/g.(3)Four different 21 bp sequence siRNA which are homologous with intI1 mRNA were designed for use to target up on the int I1 sequence,and then built siRNA-expression kit.The result revealed the si-RNA expression kit has been successfully built.(4)We electroformed the siRNA into key bacterial to realize RNAi and Quantitative Real-time PCR method was used to relatively quantify the efficiency on inhibit ion the expression of intI1.It was found that siRNA1 and si RNA2 had positive performance and siRNA2 got higher inhibition efficiency.