Molecular epidemiology of Mycobacterium tuberculosis in Gansu province of China.

BACKGROUND: Mycobacterial interspersed repetitive units-variable number tandem repeat (MIRU-VNTR) and Beijing family typing based on detecting the deletion of RD105 sequence are two common genotyping methods used to study the molecular epidemiologic characteristics of Mycobacterium (M.) tuberculosis. We collected 218 strains of M. tuberculosis between 2004 and 2006 in the Linxia Hui Autonomous Prefecture of Gansu province in Northwest China. METHODS: MIRU-VNTR analysis and Beijing family typing based on detecting the deletion of RD105 sequence were used to type the 218 strains, and their typing power was evaluated to look for practical and efficient genotyping methods suitable for the region. RESULTS: The MIRU typing yielded 115 distinct genotypes, including 98 unique isolates and 17 different clusters containing 120 isolates (55.05%); the cluster rate was 47.25%. By detecting the deletion of RD105 sequence, 188 of 218 (86.23%) isolates belonged to Beijing family. Combination of Beijing family typing and MIRU typing yielded 118 distinct patterns, including 101 unique isolates and 17 clusters containing 117 isolates (54.13%). The largest cluster contained 58 strains with MIRU genotype of 223325173533 which contained 50 strains belonging to Beijing family and 8 strains belonging to non-Beijing family. CONCLUSIONS: The Beijing family strains occupied a large proportion and the Beijing family MIRU genotype 223325173533 is a dominant strain in Linxia of Gansu. Combining detecting the deletion of RD105 and MIRU typing together provides a simple, fast, and effective method which is low in cost and might be practical and suitable for M. tuberculosis genotyping in China.

[Application of denaturing high perfomance liquid chromatography in detection of rpoB mutations associated with rifampin resistance in Mycobacterium tuberculosis].

OBJECTIVE: to explore the value of denaturing high performance liquid chromatography (DHPLC) in detection of rpoB mutations in rifampin-resistant M. tuberculosis, in order to establish a convenient and rapid approach to screening rpoB gene mutations in M. tuberculosis. METHODS: rifampin resistance-determining region of rpoB gene in M. tuberculosis was amplified by PCR and further analyzed by DHPLC to screen mutations at optimized denaturation temperature (65.4 degrees C), which utilized heteroduplex formation between wild-type and mutated DNA strands to identify mutations. The PCR products from strains with different chromatographic profiles were sequenced further to evaluate the sensitivity and specificity. RESULTS: there were 46 M. tuberculosis strains including 42 rifampin resistance strains and 4 rifampin sensitive strains. From these strains, 15 different chromatographic profiles were produced by DHPLC. Combined with the results of gene sequencing, it was shown that strains with different chromatographic profiles had distinct mutations. Except D108 and D24 which had same chromatographic profiles but different genetic polymorphisms, all other strains showed consistent chromatographic profiles with genetic polymorphisms, i.e. if they had identical chromatographic profiles, their genetic polymorphism were the same; but if they had different chromatographic profiles, their genetic polymorphism were also different. CONCLUSION: DHPLC is a simple, efficient, high-throughput and automatic method with high sensitivity and specificity, which may be useful in the rapid detection of rpoB gene mutation in M. tuberculosis.