The Research Of Detect RpoB Gene Mutation Of The Rifampin Resistant Mycobacterium Tuberculosis By Rolling Circle Amplification

PART ONEDETECTION OF THE RPOB GENE SINGLE BASEMUTATION OF MYCOBACTERIUM TUBERCULOSIS BYHYPER-BRANCHED ROLLING CIRCLE AMPLIFICATIONObjective: To establish a rapid, accurate, simple and economictechnique for the detection of Mycobacterium tuberculosis rpoB genesingle base mutation by hyper-branched rolling circle amplification.Methods:1. Through the literature review and clinical investigation,identified the rpoB gene of Mycobacterium tuberculosis in531sites ofserine mutant (C-T) for the detection target. According to the standardsequence of rpoB gene of Mycobacterium tuberculosis H37Rv standardstrain (from NCBI), design the padlock probe of detecting the genemutation, and the PCR primers for amplified clinical specimens of rpoBgene.2. Establishment and optimization of the detection of Mycobacteriumtuberculosis rpoB gene single base mutation by hyper-branched rolling circle amplification. Include the temperature of padlock probe and templateconnecting, cyclizing, and the time of amplification.3. Based on thesynthetic wild-type target sequence, the mutant target sequences, unrelatedgene sequence and without target sequence reaction systems were detected,discuss the method specificity and sensitivity.4. To detect clinicalspecimens use hyper-branched rolling circle amplification technology,verify that the value of clinical application.Results:1. The padlock probe and the corresponding templateconnecting1hours at65℃can guarantee the specificity of detection. For1hours the rolling circle amplification at37℃, can obtain obviousamplification results, and through the detection of the target sequencescontaining different concentrations of mutant samples, the minimum of thesample containing1%mutant can be detected.2. The hyper-branchedrolling circle amplification techniques were used to detect the clinicalMycobacterium tuberculosis rifampin resistant strains, rifampin sensitivestrains of Mycobacterium tuberculosis and the H37Rv strain. The testingresults in accord with the sequencing.Conclusion: We established the detection of Mycobacteriumtuberculosis rpoB gene mutation by hyper-branched rolling circleamplification technology, has the advantages of high specificity, highsensitivity, simple and easy to operate. Comparison with the traditionalmethod, this method shortened testing time, moreover, does not need special equipment and reagents, and greatly reduces the detection cost. PART TWOROLLING CIRCLE AMPLIFICATION COMBINED WITHDNA CHIP TO DETECT THE RPOB GENE MUTATION OFMYCOBACTERIUM TUBERCULOSISObjective: Combined rolling circle amplification technology andDNA chip technology, to construct a chip technology in the detection of therpoB gene mutation of Mycobacterium tuberculosis.Methods:1. Based on rpoB gene of Mycobacterium tuberculosis asthe research object, select531sites of serine mutant (C-T) for the detectionof target. According to the padlock probe of detect the gene mutation in thefirst part, Designed oligo-nucleotide probe in non site sequence of thepadlock probe combined with template, as the capture probe which coatedon the aldehyde slide.2. Through the detection of wild type and mutant typeartificial synthesis of target sequence, proved accuracy of the designedoligo-nucleotide probe and the reliability of the detection method.3. Byadopting the method to detect the clinical samples, and compared with the sample sequencing results, verify the accuracy of the method and itsclinical value.Result:1.Spotting the detection probe, the positive contrast probe andthe negative control probes on the aldehyde slide substrate, and makesample detection. The corresponding detection probes and positive controlsshowed obvious developing result, clear background, and no developmentresults in the negative control and the other sample points. It is proved thatthe design of the probe is reasonable and reliable.2. Through the detectionof different concentrations of mutant target sequence samples, to determinethe minimum concentration of the target sequence containing5fmol/Lmutant samples can be detected.3. Detect the clinical samples by adoptingthis method, results show that, with the mutant sample points and thepositive control were developing, clear background. Negative controls, thewild type, not the site mutant and non tuberculosis sequence sample pointsare not developing result. Test results are consistent with the clinicalsample sequencing proves that the method is accurate and reliable.Conclusion: This study successfully established technology combinedthe RCA and the DNA chip in the detection of the rpoB gene single basemutation of rifampin-resistant Mycobacterium tuberculosis. This techniquehas high specificity, high sensitivity, no need special instrument, simpleoperation, can be used for the detection of clinical samples.