Construction of standard plasmids for detecting Salmonella in meat products with real-time fluorescence quantitative polymerase chain reaction / 上海预防医学

Objective To construct standard plasmids for detecting Salmonella in meat products with real-time fluorescence quantitative polymerase chain reaction(PCR).Methods Primers directed at Salmonella invA gene were designed.Specific fragments were amplified and cloned into plasmid vectors to construct recombinant plasmids.The constructed recombinant plasmids were used as standards for implementing the real-time fluorescence quantitative PCR after optimization, establishing standard curves and examining the sensitivity and stability of these standards.Results The plasmid standard containing the invA gene in Salmonella was successfully constructed.The cycle threshold value(Ct value) of the standard curve established by means of the plasmid standard and the number of template copies exhibited good linear relationship(r2=0.9979).The minimum that could be detected by means of this method was 10 copies/reaction.The standard plasmid was proved to have good stability.This standard plasmid was used to detect Salmonella in meat products.After two hours enrichment, sample detection could be completed within seven hours.Conclusion The constructed plasmid standard can be used for detecting Salmonella in meat products by means of the fluorescence quantitative PCR, which can provide reliable reference bases for examining quality of relevant experiments.

Establishment and preliminary application of real time PCR assay for quantitative detection of CRLF2 / 国际检验医学杂志

Objective To establish a real‐time quantitative PCR method for the detection of cytokine receptor‐like factor 2 (CRLF2) expression .Methods Specific primers amplification target gene CRLF2 and housekeeping genes ABL were designed ,the purified PCR products were performed the TA cloning .After bacterial colony PCR screening and sequencing ,then the recombinant plasmids DNA was extracted and measured by using UV spectrophotometer and converted to copies/mL concentration .Finally it was diluted for preparing the plasmid standard substance ,then the standard curve was drawn for observing the sensitivity and linear rang ,meanwhile the stability of the plasmid DNA was evaluated .This method was initially applied to detect the CRLF2 level of bone marrow mononuclear cells in 10 cases of healthy children and 10 cases of newly diagnosed acute lymphoblastic leukemia (ALL) .Results CRLF2 PCR product had a single specific melting curve;the linear detection range of the standard substance was 103 - 108 copies /ml;the plasmid standard substance by freeze‐thawing for 3 times remained stable;the CRLF2 level of clinical sample was within the linear detection range of standard substance .Conclusion The real‐time quantitative PCR method for CRLF2 established by our laboratory has good specificity ,linearity range and stability ,which can be applied to the quantitative detection of CRLF2 gene in clinical ALL children .