Preparation Of Diarrheogenic Escherichia Coli Plasmid DNA Qualitative Reference Material And Its Application In The Detection Of Lycium Barbarum

Nucleic acid standard sample refers to the standard sample used in nucleic acid amplification technology,which plays an important role in promoting the standardization of detection methods of pathogenic nucleic acid amplification and improving the consistency and accuracy of detection results.In China,the development of nucleic acid standard samples is in the beginning,the quantity and species are far from meeting the needs of the current market and supervision and inspection,and there is a lack of plasmid qualitative standard samples related to virulence gene monitoring of Escherichia coli.In this thesis,the amplification products of the virulence genes esc V,stx2,hly A,rfb,uid and eae A were obtained by PCR.The target fragment was purified by agarose gel,and the purified esc V,stx2,hly A,rfb,uid,stx2,and eae A target fragments were cloned into the vector p LB-simple Vector to construct esc V,stx2,hly A,rfb,uid,stx2,eae A multiple recombinant plasmids.Recombinant plasmids were verified by colony PCR and sequencing.Colony PCR and sequencing proved that the recombinant plasmid was successfully constructed.The results of colony PCR showed that the success rate of transformation was 100%.The sequencing results were compared with the published gene sequences on Gen Bank.The results showed that the comparison rate of uid and other sequencing results was 99.6% and 100% respectively,indicating that the recombinant plasmids were constructed successfully.The detection limit and specificity of the plasmid standard were tested by the established PCR reaction.The results showed that the detection sensitivity could reach pg level,and the plasmid standard had specificity.The recombinant plasmids that had been successfully transformed were transferred to 15 generations sequencing.The results of 15 generations sequencing showed that the constructed plasmid standard was stable.The constructed recombinant plasmids esc V,stx2 and hly A were prepared by freeze-drying powder and used as templates for detection limit experiments.The results showed that the detection limits were 3.93×10⁶ copies·?L-1,2.41×10⁵ copies·?L^-1 and 2.14×10⁵ copies·?L^-1,respectively.The homogeneity of esc V,stx2 and hly A plasmid standard freeze-dried powder samples was tested.The results showed that there was no significant difference between freeze-dried powder and the uniformity of samples was good;Take 30 tubes of esc V,stx2 and hly A plasmid standard freeze-dried powder samples at random and store them at 25?,4? and-20?,and take three samples at fixed time for long-term stability test.The results of concentration determination showed that the plasmid standard freeze-dried powder had good stability.The properties of freeze-dried powder were determined by PCR.The PCR results showed that the properties of freeze-dried powder were stable at three temperatures.The constructed plasmid standard of common virulence genes esc V,stx2,hly A of E.coli was used as a positive control for the detection of 34 batches of L.barbarum samples.Results showed that esc V and stx2 were not detected in the 34 batches of samples,the detection rate was 0.Hly A was detected in 3 batches of L.barbarum samples,and the detection rate was 8.82%.Three batches of L.barbarum samples with positive PCR results were selected for traditional microbial isolation and identification,and no E.coli was detected.The bacterial enrichment solution used in the microbial detection method did not specifically target the bacterial growth.In the microbial contaminated environment,the target pathogenic bacteria are likely to be inhibited from growing,which greatly reduce the detection rate.This study took foodborne microbial E.coli as the research object,constructed plasmid DNA standards of common virulence genes in E.coli and applied them to L.barbarum for detection.