Preparation Of Chip For The Detection Of The Main Resistance Genes In Mycobacterium Tuberculosis Isolates

Objective:Detecting rapidly the hot mutations of drug resistance gene in Mycobacterium tuberculosis using oligonucleotide gene chips in order to research and accurate method in gene diagnosis of Mycobacterium tuberculosis.Methods and Result:Part one:cloning and identification of gene chip for the detection target genes of mycobacterium tuberculosis isolates1.Samples of drug resistance Mycobacterium tuberculosis were collected and genomic DNAs were extracted.2.After DNA sequences of drug resistance gene in Mycobacterium tuberculosis ware analysed using gene bank,Probes and Primer were synthesized.3.Seven target gene fragments were obtained by PCR amplification,and recombinant plasmids of target gene were obtained by pMD18-T cloning and selection,at last identificat then by digestion and PCR technique.4.The further analysis from multiple alignment and Blast indicated that,the target genes cloned and identified are highly conserved in target pathogen,which provide reliable materials for the Mycobacterium tuberculosis gene chip construction.Part two:construction of gene chip for the detection of mycobacterium tuberculosis isolatesGene chip for the detection target genes of mycobacterium tuberculosis isolates,methods for probe preparation and hybridization temperature of target genes as well as specification were investigated in this study.And the target genes of gene chip and their concentration,the controling methods of the related chip quality are determined.1.Preparation of targeet genes and genes for loacation control Used the recombined plasmids as templates,and the target genes were amplified by PCR,then were purified by isopropanol precipitation method.The fluorescence Cy3 marked primers are used for PCR amplification of Exon 21 and Exon22 again. The concentration and purity meet the requirements of the chip production.2.Oligonucleotide probes were design and synthesized according to the common mutation of the target genes.The probes are 5'-marked with a concentration around 121 to 748ng/μL.3.The probes were typed on a aldehydlized substrate plate in a certain sequence,then maked up a chip.4.A hybridization was carried out using fluorescence marked target genes with the chip.After developing and analysis,the results were confirmed.5.Through this study,a optimum concentration,300ng/μL of the target genes,was confimed.The sensitivity of chip system is 3pg/μL.A number of target genes with strong hybridization signals and very well hybridization specificity were screened out,and 1h of pre-hybridization time and 5h of hybridization time were established as well.Conclusion:The chip product of this research can be used in examination of the drug resistance gene in Mycobacterium tuberculosis,and which gives a considerable specificity and sensitivity,so it coud be applicated in clinic examination of the drug resistance gene in Mycobacterium tuberculosis.This application is identified by its speedness,conveniency and high sensitivity.