Typing MLST Genotyping Research And Standardization MLVA Chinese Mycobacterium Tuberculosis

The study of tuberculosis (TB) molecular epidemiology of is very impoternt use for exploring TB transmission mechanism, epidemic regularity, etc. The core technology of molecular epidemiology is genotyping methods. At present, IS6110-RFLP (insert sequence6110restriction fragment length polymorphism), Spoligotyping (spacer oligonucleotide typing), and MLVA [Multiple Loci VNTR (varisble number tandem repeat) Analysis] are the commom methods in the tuberculosis molacular epidemiological research, and have a very important role for etiology, epidemiology, and the infection mechanism research.To establish the standard MLVA methods used in genotyping and molecular epidemiology surveillance in China,128M. tuberculosis clinical isolates were typed with46VNTR loci. The loci were filtered by PCR result firstly,13loci were excluded because they had no PCR result, such as Qub11b, and3loci were excluded because of the complicated PCR results, such as Rv1895; and then duing to the discrimination index (Hunter-Gaston index, HG index),16loci (MLVA-16) including Mtub21、 MIRU26、Qub26、Qub11-a、ETRE、Mtub04、MIRU10、MIRU16、MIRU40、 Mtub30、ETRA、MIRU39、Mtub38、Mtub3、ETR-F、and ETRD were comfirmed to be the chosed loci in this standard protocol.12MIRU (Old12),12MIRU plus3ETR (Old12+ETR),15loci-combination and24loci-combinantion presented by Supply in2006were the common used loci-combination in global. The discrimination indexes of those loci-combinations were compared with that of MLVA-16. The highest discrimination indexes were24loci-combination and MLVA-16(0.999),15loci-combination presented by Supply in2006was approximately, but the discrimination indexes of O1d12and O1d12+ETR were much lower. 128strains including126clinical isolates, H37Rv and BCG were genotyping by MLVA-16, IS6110-RFLP and Spoligotyping. The protocols were refered to the published reference. Compare the HG index of the three methods in all the strains and the Beijing family strains that was the majority in Chinese M. tuberculosis strains, was the bast way to undersdand the discrimination power of those methods.By means of MLVA-16,128M. tuberculosis were divided into11groups. There were120genotypes, including115orphern types, the HG index was0.999(CI0.998-0.999);102Beijing family strains were divided into94genotypes, including89orphern types. The MLVA result was submitted into MIRU-VNTRplus webnet (http://www.miru-vntrplus.org/MIRU/aboutus.faces), and compared with that of other countries. Most of those strains isolated in China were congregated with the Beijing family strains in webnet database, and others were separated distribute.In this study, IS6110-RFLP has the highest discrimination index not noly in128strains but in Beijing family strains. The discrimination power of MLVA-16was much similar with IS6110-RFLP, and the consistency was91.1%, so MLVA-16could be the first line genotyping method in Chinese M.tuberculosis molecular epidemiology research, for many unoverstepped shortcomings of IS6110-RFLP. Spoligotyping has the lowest discrimination power.A CAS strain has been found in Xizang province, and2CAS-like strains were been found in Xinjiang province. The MLVA-16results of those3strains were simlar too, but the IS6110-RFLP result was much different with each other, so whether those2CAS-like strains belonged to CAS family need more research to identify. The CAS family strain may be induced by the floating population, such as travel people, for CAS family was ususally distributed in India area. Single nucleotide polymorphism (SNP) is a very important genetic marker, which multiple locus sequence typing (MLST) analysis based on was becoming to be a new and useful genotyping method. MLST involves obtaining the sequences of internal fragments of seven house-keeping genes for each strain of a particular species. The MLST databases are currently hosted on two main web servers located at Imperial College Londen (http://www.mlst.net) and Oxford University (http://pubmlst.org). Those website contain lots of data of many bacterial such as Neisseria meningitides, Staphyloccocus aureus Rosenbach, Streptococcus pneumoniae, Campylobacter jejuni, Helicobacter pylori, etc, and fungal like Blastomyces albicans, but no M.tuberculosis still.This study compared the whole genome of CCDC05079, CCDC05180(provided by TB Laboratory of National Institute for Communicable Disease Control and Prevention (ICDC), Chinese Center for Disease Control and Prevention (China CDC), and H37Rv published in NCBI, chosed the housekeeping genes which had difference, and analyzed the discrimination power of each housekeeping genes, then confirmed seven genes(rmlC、mpt53、panD、gcvH、recX、mprA and ideR). The seven gene sequences of49M. tuberculosis clinical isolates were analyzed by MEGA4software, and the evolution tree was drawed by NJ method.49strains were devided into2groups by SNP(rmlC101G→C、rmlC345A→C, amino acids mutation RmlC34G→A、RmlC115E→D) in rmlC, which encode dTDP-4-krto-6-deoxyglucose3,5-epimerase, taking part in the metabolism of dTDP-rhamnose. Though dTDP-rhamnose was very impotant in sustaining cellular wall and bacili growth, the enzyme participated in the metabolism of rmlC were importante. The difference in rmlC maybe reveals the potential differenc in growth of the two group strains.Some virulence related genes were used in MLST analysis in this study, including panD、ideR、mprA, so genotyping result could have some relationship with the virulence of strains. Then MLST was not only useful for the study on the disease transmition and infectius source detection, but significant for the relationship of the genotyping with virulence of M. tuberculosis.Otherwise, the sequence of housekeeping gene were compared and assigned a distinct allele number or STs (sequence typing). The BURST (Based upon related sequence types) was used for analysis of genetypes, and49strains were devided into40STs, including the main popular group:the clone centered by ST9and ST2. It was found62SNP loci in the7housekeeping gene of49strains, which became the basement of MLST database of M. tuberculosis in China.