| Infection of Mycobacterium tuberculosis (MTB) is the most significant cause of death in infecious agents, which leads to death of 2 million annually. Since the middle of 1980's, the epidemiological situation of tuberculosis has been becoming more and more serious due to the ignoring of tuberculosis, the prevalence of AIDS, the emergence of drug-resistant strains of MTB and the increase of folating population,and so on. As the incidence of tuberculosis has increased, there has been a corresponding rise in the incidence of drug-resistant strains of MTB. Rifarmpicin (RFP) is one of the most potent antituberculosis drugs; therefore, resistance to RFP often results in high clinical relapse rate, particularly if RFP resistance is associated with resistance to other antituberculosis drugs, such as Isoniazid (INH). Moreover, more than 90% of RFP-resistant isolates are also resistant to isoniazid; therefore, detection of RFP resistance could also identify MDR strains.Early diagnosis of the disease and rapid identification of resistance to primary antituberculosis agents are essential for efficient treatment and control of multidrug-resistant strains.To investigate situation of rpoB gene mutations in MTB, and identify the characteristic and the distribution of the mutation, clinical isolates of MTB from the patients with tuberculosis in Xizang, Hunan, Henan, Sichuan, Fujian, Anhui, and Shanxi provinces of China were collected and identified by means of biochemistry reaction method. The drug-susceptibility of the isolates was determinated by proportional drug susceptibility testing. Then the rpoB gene fragment was amplified by PCR and the mutations in the rpoBgene fragment were detected by DNA sequencing. Based on the sequencing result, the mutation was analyzed, including the rate of every mutation site, the kinds of the rpoB gene mutation in MTB and the correlation between the rpoB gene mutation and drug-resistance. Then to select distinctive mutation site by which the oligonucleotide probe was designed oligonucleotide probe reverse dot blot hybridization (RDH) was established based on PCR to detect the mutation of rpoB gene rapidly. Moreover, the data from all of the tests were combined for analysis using SPSS 11.0 software and investigated that whether it could be used as the clinical screening method of RFP- resistane or not, and assessed its value of application.536 clinical isolates of MTB from the patients with tuberculosis were identified by means of biochemistry reaction method, of which 482 (89.93%) was MTB strains, 19 (3.54%) was M.bovis strains, 33 (6.16%) was Non-tuberculosis Mycobacterium(NTM),2 (0.37%) was MTB and NTM by which the patient was co-infected.The drug-susceptibility of 482 clinical isolates of MTB were determinated by proportional drug susceptibility testing. The results showed that 126 strains (26.14%) were sensitive to all 5 drugs, 356 strains (75.52%) were resistance, in which 88 strains (18.26%) were resistant to RFP only and 268 strains (55.60%) were MDR strains.300 MTB clinical isolates including 119 RFP-sensitive strains and 181 RFP-resistance strains were selected to investigate the the mutations in the rpoB gene of MTB. Mutations were found in 166 of 181 RFP-resistant strains in the fragment amplificated by DNA sequencing, the mutation rate is 91.71%. 531 site, 526 site and 516 site were the most common sites to mutate, mutation rate of 531 site was 53.61%, of 526 site was 27.71%, of 516 site was 9.64%, and of 511 site was 6.02%. The sum of mutation frequency of such four mutation sites was 96.99% in all mutation sites. Combinative mutations rate was 9.04% (15/166). The mutation of one DNA code insertion was found in 1 strain, and oligonucleotide or DNA code delection was found in 5. No mutation was found in 67 completely sensitive strains and the mutation of 511 site and 526 site was found respectively in 2 strains of 52 sensitive strains only to RFP.The mutation of rpoB gene in 300 strains which the mutation had beenanalyzed by DNA sequencing was detected by means of oligonucleotide probe reverse dot blot hybridization based on PCR. The results showed that no mutation was found in 67 completely sensitive strains and the mutation was found in 2 strains of 52 sensitive strains only to RFP. Within 181 RFP-resistant strains 165 strains were detected to be mutation strains, no mutation was found in 16 strains. Compared with DNA sequencing, the sensitivity and specificity of RDH was 97.62% and 97.73% respectively, the concordance with DNA sequencing of MTB was 97.67%,there was no significant difference between RDH and DNA sequencing by Chi-square statistics test﹙P>0.05﹚.In conclusion, the mutations of rpoB gene in this research are consistent to the other research repots from internal and external coutry. 531, 526, 516 and 511 sites are the most common positions of mutation, and the proportion of them are 53.61%, 27.71%, 9.64%, and 6.02%, reseptively. Meanwhile, mutations also exist in some sporadic sites, such as 515, 513, 510, 509, 512, 514, 522 and so on. There are combinative mutations and delection and insertion of basic group. Moreover, reverse dot blot hybridization based on PCR can be used to detect gene multi-mutations at one time, and its results can be justified directly with naked eyes easily. The coincidence of RDH and DNA sequencing can achieve 97.67%, there is no significant difference between the two methods by Chi-square statistics test (P>0.05). This method is so rapid, sensitive, and simple that can be applied for screening the RFP-resistance of MTB clinical isolates. Furthermore, as the technical foundation of geng chip, RDH has extensive perspective of clinical rapid diagnosis of RFP-resistance.
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