| In the process of storage and preparation of traditional Chinese medicine,pathogenic bacteria pollution will occur.Salmonella is one of the common pathogenic bacteria of food borne microorganisms.After infection,it will lead to poisoning,vomiting,diarrhea and other diseases.Therefore,the detection of Salmonella is necessary and strict,while the traditional microbiological detection method is cumbersome and low sensitivity,which fails to meet the needs of rapid detection.Therefore,using modern molecular biology technology to improve the specificity and sensitivity of detection has become a hot topic.In this thesis,four pairs of primers were designed and synthesized for Salmonella virulence genes inv A,fimI,mgtC and fimA according to the national standard and the common virulence genes of Salmonella in the literature.The genomic DNA of Salmonella typhimurium CMCC 50071,Salmonella typhimurium CMCC 50115,Salmonella typhimurium ATCC 14028,Salmonella enteritidis CICC 21482,Salmonella CMCC 50956,Salmonella CMCC 50957,Salmonella CMCC 50958,and Salmonella CMCC 50959 were extracted.The whole genomic DNA of Salmonella was used as template for PCR amplification inv A,fimI,mgtC and fimA PCR amplification products were obtained,and purified inv A,fimI,mgtC and fimA were purified by agarose gel,and the purified target fragments were joined and transformed by inv A,fimI,fimA and gamma respectively,and cloned into vector vector to construct recombinant plasmids containing the fragments of E.The recombinant plasmids were verified,and the transformed plasmids were extracted and preserved,then lyophilized to make freeze-dried powder,which was used to detect the standard of Salmonella.Results the recombinant plasmids of virulence gene inv A,fimi and mgtc were successfully constructed,and tested for homogeneity,randomness and stability.The results showed that the concentration,purity and stability were up to the requirements,so as to complete the preparation of qualitative standard of virulence gene plasmids of Salmonella.This thesis,81 batches of four Huaiyao from Jiaozuo,Henan Province,including 23 batches of Huaihua chrysanthemum,18 batches of Huaishan yam,20 batches of huaidihuang,20 batches of huainiuxi,were used to extract the whole genome DNA of 81 batches of four Huaiyao,and PCR amplification was carried out.The inv A and fimI plasmid DNA standards of common virulence genes of Salmonella were used as positive control and sterile water as negative control,which were used for the detection of 81 batches of Huaiyao samples The results showed that fimI was not detected in 81 batches of samples,the detection rate was 0,inv A was detected in 2 batches of chrysanthemum samples,the detection rate was 8.69%,and inv A was detected in 1 batch of Rehmannia samples,the detection rate was 5.00%.The polluted chrysanthemum and Rehmannia samples were selected and detected by traditional microbial detection methods.After enrichment culture,the eighth batch of samples were on the selective color medium plate The second Rehmannia sample and the 15 th batch of chrysanthemum sample did not grow colonies,and the 7th batch of chrysanthemum grew typical colony morphology of Salmonella on the selective plate.The purified single colony was selected for PCR amplification of 16 S r RNA and sequencing comparison,so as to complete the research and application of Salmonella in 81 batches of Huai medicine.In contrast,PCR detection rate is higher and time-consuming.Food borne Salmonella was taken as the research object,and the plasmid DNA standard of common virulence genes in Salmonella was constructed and used in traditional Chinese medicine for detection,which provided a clinically applicable technology and plasmid standard,and also provided help for clinical traceability. |
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