16SrDNA And CFP10 Gene Fluorescence Quantitative PCR Application In The Diagnosis Of Spinal Tuberculosis

Objective Explore the real-time fluorescent quantitative PCR technology applied in early diagnosis of spinal tuberculosisMethod Choose 16 Sr DNA and CFP10 two gene as a target of this experiment, designed two pairs of specific primers, respectively using SYBR Green I method(fluorescent dye method) to establish a fluorescence quantitative PCR reaction system, based on the genetic database provided by the conservative sequence of two genes, gene were obtained using artificial chemical synthesis method, construct the recombinant plasmid body p GH- 16 srdna and p GH- CFP10, and the recombinant plasmid body to identify the enzyme digestion and sequence determination, the successful build positive standard, and making the standard curve and the curve equation, finally using this method in 20 cases of patients with clinical diagnosis of spinal tuberculosis plasma specimens and 20 cases of not spinal tuberculosis patients plasma samples for testing and quantitative.Results Two standard curve was good linear relationship, the correlation coefficient of 0.999 and 0.998 respectively, high sensitivity, respectively is 1.48x101copies/ul and 2.67x101copies/ul, and good specificity, dissolve two present single peak curve. Serum in patients with spinal tuberculosis in 20 cases of clinical MTB- DNA test results are all positive, 20 cases of not spinal tuberculosis patients of serum MTB- 19 negative in DNA detection, 1 case was positive, and testing a sample time is only 2h.Conclusion Successfully established with the target genes of 16 sr DNA and CFP10 spinal TB fluorescence quantitative PCR detection method, can be used for clinical patients with spinal tuberculosis pathogen detection and quantitative.