Genome-wide Screening And Applying DNA Bar-coding Genes To Reveal Molecular Evolution Of Mycobacteria

In recent years, with the frequent movement of the population, the multi-drugresistant(MDR) strains of Mycobacterium and the prevalence of humanimmunodeficiency virus (HIV) infection, the incidence of TB infection was still veryhigh. Meanwhile, more and more environmental non-tuberculosis mycobacteria(NTM)infection cases were reported. The manifestations were too similar between TBinfection and NTM infection to diagnosis and most NTM were naturally resistant toanti-TB drugs. Therefore, it was urgent to monitor the molecular epidemiology ofmycobacterial and find a scientific and effective typing method. The scientificallyaccurate genotyping methods of mycobacterial had very important theoreticalsignificance and application value for the study of dynamics of the spread ofmycobacterial and accurate identification of the pathogen and real-time monitoring.Our study would display the genome by barcode-like visual annotationtechnology to screen the polymorphism and relatively conservative gene as a barcodefor molecular typing. We set W(Windows)=1000bp,k(k-mer)=4.We screened threegenes by this technology which are Rv0279c、Rv3508、Rv3514respectively. We used16S rRNA and these three genes together as DNA barcode to reveal molecular typingof mycobacterium tuberculosis, including12mycobacterium tuberculosis complexstrains,3non-tuberculosis mycobacteria strains and2mycobacterium leprea strains.Then, we used Mega5.05software to build evolutionary tree and Pfam_Scan databaseto study the functions of three genes. We have collected5clinical specimens oftuberculosis which were from the Infectious Diseases Hospital of Changchun. Weinvestigated the five clinical specimens for DNA extraction and PCR and sequencingof the three genes,thus calculated the mutation rate. From the results, we could conclude that these three genes had relativelyconservative area which had been verified in clinical strains. At the same time, thethree genes were rich in variation district. The three genes and16S rRNA as genebarcode could distinguish the17strains of mycobacteria clearly. The proteinsencoding by three genes are associated with virulence, belonged to the the PE/PPEfamily protein. The Rv0279c involved in regulation of the host iron metabolism. TheRv3508involved in oxidative stress. Rv3514is one of the members of the cell surfacesecreted protein of ESX family. Screened three genes from the genome-wide mass ofinformation applied new vision and understanding for us to study the moleculartyping of mycobacteria. The high polymorphism resulted from mutation rate wasbenefited for genotyping.