The Sequence Analysis Of 16S RRNA Gene And 16S-23S RRNA Gene Internal Transcribed Spacer Of Interspecies And Intraspecies Of Mycobacteria

Objects 1.Establish the 16S rRNA gene and 16S-23S rRNA ITS sequence analysis methods to identify the Interspecies and Intraspecies of Mycobacteria,while understand the value of 16S rRNA gene and 16S-23S rRNA ITS sequence on the identification of Mycobacteria.2.Master the gene sequence of 16S rRNA gene 5' and 16S-23S rRNA ITS of 123 Mycobacteria(total of 213 strains),the sequence information could be the supplement of the Gene Database.Methods The 16S rRNA gene 5' and 16S-23S rRNA ITS of 123 Mycobacteria(a total of 213 strains)were amplified and sequenced respectively by PCR amplification according to the designed genus-specific primers of Mycobacteria.The 16S rRNA gene 5' and 16S-23S rRNA ITS sequence of Mycobacteria were homologous multi-sequence compared by Clustalx 1.83 and we constructed the Cluster Analysis tree spectrum by the Neighbour joining way (N-J way),and obtained the similarity percentage of interspecies and intraspecies of Mycobacteria with DNA STAR Megalign software.Results1.Completed the sequencing of 16S rRNA gene of 119 Mycobacteria species,including 209 strains,and the sequencing of 16S-23S rRNA of 123 Mycobacteria species,including 213 strains.2.During the identification of interspecies we found 3 members of MAC's 16S rRNA gene,16S-23S rRNA ITS was respectively identical;16S rRNA gene and 16S-23S rRNA ITS were respectively identical among 5 Mycobacteria tuberculosis Complex members, M.marinum and M.ulcerans.The Mycobacteria above could not be identified by two target genes.Moreover,Mycobacterium tuberculosis 95052(ATCC35810)and the other five members of MTC;M.gastri and M.kansasii;M.farcinogenes and M.senegalense;M.chelonae and M.abscessus;M.porcinum.and M.boennickei;M.vaccae(95050)and M.austroafricanum; M.peregrinum and M.septicum;M.vanbaaIenii and M.vaccae;M.frederiksbergense and M.gilvum;M.murale and M.tokaiense could not be identified by 16S rRNA gene,but could be identified by 16S-23S rRNA ITS.M.paratuberculosis and MAC;M.chitae,M.agri,and 5 MTC;M.aurum and M.tokaiense; M.thermoresistibile and M.parafortuitum;M.vanbaalenii and M.austroafricanum; M.flavescens and M.pulveris;M.obuense and M.vaccae;M.duvalii and M.porcinum; M.hassiacum and M.pyvenivorans could not be identified by 16S-23S rRNA ITS but could be identified by 16S rRNA gene.3.During the analyzing of intraspecies of mycobacteria(7 species total of 97 strains)we found that 13 M.phlei could be divided into 3 genotypes by 16S rRNA gene,and 18 M.fortuitum could be divided into 6 genotypes,17 M.smegmatis could be divided into 4 genotypes,8 M.gordonae could be divided into 3 genotypes,9 M.chelonae could be divided into 3 genotypes,15 M.kansasii could be divided into 2 genotypes,and 17 M.farcinogenes only have 1 genotype.The strains above could be divided into 3,15,7,3,4,3,5 genotypes by16S-23S rRNA ITS.Conclusions 1.16S rRNA gene and 16S-23S rRNA ITS sequence analysis is a good method for identifing Mycobacterium species.When combined with the two target gene we can identify most of the species in very short time.2.The polymorphism of 16S-23S rRNA ITS among intraspecies of Mycobacterium is higher than that of the 16S rRNA gene,Some strains could not diffemted by 16S rRNA gene were differented by 16S-23S rRNA ITS.16S-23S rRNA ITS is more suitable for classification and identification for intraspecies of mycobacteria.3.The gene sequences of all strains could be the standard reference sequence in clinical isolates identification,and be the supplement of gene database.Lay a good foundation for further designation of Mycobacterium species probe and the research of gene chip.