Establisment And Evaluation Of Taqman PCR Detect Method Of Mycobacterium Tuberculosis Complex And MLVA Genotyping Study On MTBC Isolated From Deer In Jilin Province

Deer tuberculosis (TB) is a chronic bacterial disease of animals and humans caused by Mycobacterium bovis, which is caused by M. tuberculosis and M. bovis. Since the world's population of feeding deer is now up to 5 million, there has been an increasing awareness of the potential threat posed by TB to domesticated deer, and Deer TB is a source of infection for human TB. It is important to identify isolates, genotyping and Drug Resistance of the MTC for epidemiologic and public health considerations and M. tuberculosis to optimize treatment.For establishment a method of rapid specific detection TB, the real-time PCR assays for specific detection of pathogenicity Mycobacterium tuberculosis, Mycobacterium tuberculosis hominis and Mycobacterium bovis were developed. The standard recombinant plasmids pEASE-MTC, pEASE-B1, pEASE-M1 were constructed as reference standards used in absolute quantification assaying. The CT values and ln(x) were highly correlated (MTC R2=0.9998, B1 R2=0.997, M1 R2=0.9991). The dynamic range of measurement is 103-108 Copies/μL. The assays have positive result with Standard quality control strains, the detection result of BCG and microorganisms is negative, and the assays could detect single bacterial cells. Upon detecting 45 clinical samples that were positive in PPD test, the results of Taqman-PCR showed 36 to be positive. However, there were 7 positive samples in Taqman-PCR assay of 50 samples, while they showed negative results in PPD test. The result showed a method of rapid specific identification of TB had been successfully established, and this method might be of great significance in the early diagnosis and rapid detection of TB.In order to investigate the prevalence of deer Tuberculosis in Jilin province of China, a total of 1856 blood samples from 20 deer farms were examined by ELISA which used Ag85-East6-mpt70 protein as the diagnostic antigen. The result of prevalence survey showed deer tuberculosis exists in Jilin Province at a positive rate of 17.83%, Jilin city had the highest positive rate of 23.22%; the lowest positive rate was 11.33% in the Songyuan region. A significant difference was seen in six geographical areas, different gender and different age groups. The results also showed that prevention and control of the transmission of deer tuberculosis can be enhanced by the efforts of epidemic prevention departments.60 strains of acid-fast positive bacilli were cultured from 138 Taqman PCR-detected positive tissue samples by 7H9 medium, including 48 strains of MTBC (43 strains of M.bovis and 5 strains of M.tuberculosis),5 strains of M.bovis which were cultured from ELISA negative samples, and 5 strains of M. tuberculosis which were cultured from ELISA positive samples. The culture result showed Taqman PCR is more specificity than ELISA.Let us assess the diversity of 24 MLVA loci in 48 stains of MTBC isolated from deer, and discuss the characteristic of the Mycobacterum tuberculosis from Deer. The genotypic characteristics of MTBC in Jilin province were investigated using MLVA genotyping method. The MLVA loci were amplified by PCR which used extracted genomic DNA of MTBC isolates as template, the amplification products were analysis by CE. The number of copies of tandem repeats of 24 MLVA loci were confirmed based on the length of PCR amplification products. The diversity of 24 MLVA loci was assessed by the number of repeated sequences. The discriminatory power of different MLVA loci complex was assessed by Hunter-Gaston index(HGDI). The results of diversity analysis showed:QUB11b was highly discriminative(h≥0.6), ETRA, MIRU4, MIRU23, MIRU31, MIRU39, MIRU40, QUB26, Mtub4 and Mtub21 loci were moderately discriminative (0.6>h>0.3), MIRU2, Mtub30 and Mtub34 were poorly discriminative(h≤0.3), while the other loci had no diversity.13 diversity loci showed similar discrimination with all 24 MLVA loci, and 7 different genotypes were identified with a genotypic diversity indice of 0.903. The set of the 6 loci with the most polymorphism (h>0.5; ETRA, MIRU4, QUB11b, QUB26, Mtub4, Mtub21) also had the same power as all 24 MLVA loci. The 6 diversity MIRUs loci resolved the 48 isolates into 11 genotypes, with a genotypicdiversity indice of 0.890.4 diversity Mtubs loci resolved the 48 isolates into 9 genotypes, with a genotypicdiversity indice of 0.857. The result of this study indicated that the MTBC from Deer in JiLin province were different from other regions, and MLVA tecnique is very useful for the epidemiological investigation of Deer tuberculosis, so it will exert important function in the epidemiological investigation of Deer tuberculosis.A study on Mycobacterium tuberculosis, which clinically isolated the molecular resistance mechanism of RFP, SM and INH, analysed tuberculosis drug susceptibility of 48 strains MTBC by MABA assay. It selected one INH and four SM strains, then amplified and sequenced the rpsL gene fragments(830bp)and KatG gene fragments(626bp) of the 5 strains. The result showed that 1 INH resistant strains eperienced mutation in 315 site of KatG gene(AGC to ACC), while three SM strains had mutation in 43 site of rpsL gene(AAG to AGG) and the other one SM resistant strains did not show any occurrence of mutation. The detection of DR-TB cases indicated that epidemic spreading of DR-TB might exist within a certain scope to some extent in deer farms of Jilin province, so it is necessary to reinforce the surveillance of drug resistance, especially at the molecular level, in order to diagnose the expansion of mycobacterium tuberculosis at an early stage, which is also important to the ability to control the epidemic situation of DR-TB.