| Tuberculosis is an infective disease that has done harm to human being, health seriously for a long time. For recent ten years late, there is a increase tend at the incidence of tuberculosis in many countries, tuberculosis has done seriously to human being, s health and life. In global infective illness, tuberculosis is the first death reason of adult. Reported by WTO, in 1997 there are about 7000 thousand new emergence tuberculosis patients in the world, nearly about 3000 thousand people died of tuberculosis. There are about 3.3 hundred million people who have been infected Mycobacterium tuberculosis in China, and there are about 5900 thousand TB patient in China, nearly 25 percent of the world. Every year died of tuberculosis people were 250 thousand. It is very important that strengthen the tuberculosis therapy. Anti-tuberculosis chemistry therapy drug is import to tuberculosis. The patient can recover thoroughly by reasonable chemistry therapy .The emergence of drug-resistance and multiple drug-resistance (MDR) mycobacterium tuberculosis is the key reason of controlling lung tuberculosis. It is reported by the forth china drug resistance of mycobacterium tuberculosis (MTB) epidemiological survey that china drug resistance of Mycobacterium tuberculosis is following: The resistant rate to any drug was 27.8﹪in all MTB isolates, 18.6﹪in new case, and 46.5﹪in previously treaded case, Multiple drug -resistant rate was 10.7﹪. It showed that resistance tuberculosis disease was 30﹪percent of total tuberculosis, multiple drug-resistance is very seriously. It suggest us it is very important that we search rapid detection the drug-resistance MTB method in treating TB. Searched the drug-resistance strains can increase the recovery rate of treating tuberculosis. Drug-resistant of tuberculosis is a waiting for resolution question in clinical works. Rifampin is a key drug of anti-tuberculosis, since 1993 domestic and foreign country scholars have researched the molecular basis of resistance to rifampin, rifampin-resistant strains of Mycobacterium tuberculosis was due to mutations in rpoB gene encoding theβ-subunit of the DNA-dependent RNA polymerase. The mutations appeared in rpoB gene encoding (81-base pair)27 amino acids region at codon position from 507 to 533.The strains can was identified drug-resistance to rifampin by analysis drug-resistance genotypes. The experiment analyzed the rpoB gene mutations characterization of 20 rifampin drug-resistance Mycobacterium tuberculosis strains in changchun city, questioned the possibility by polymerase chain reaction and direct sequencing (PCR-DS) method. The experiment method: 1. materials:MTB (human type) international standard strain H37RV, rifampin drug-resistant MTB international standard strain ,atypical MTB (bird type) international standard strain, control strain Streptococcus pneumonias, there are 20 rifampin drug-resistant strains and 5 rifampin-sensitive strains fromchangchun tuberculosis hospital patient sputum examples.2.Ready for DNA examples: The genomic DNA from purchased strains and control strain was obtained by boiling method, The DNA from clinical sputum examples was obtained by TritonX-100 lysis boiling method.3.Using above DNA as template, upstream and downstream primers was amplified by polymerase strain reaction. The amplification reaction volume was 50μl, contained 10×buffer 5μl; dNTP concentration was 200mmol/L, took5μl; primer concentration was 10pmol/l, took 5μl; template DNA 2μl; TaqDNA polymerase 0.25μl; took ddH2O 33.75ml. The cycling reaction parameters included: initial denaturation at 95℃for five minutes, continued denaturation for one minute, annealing at 56℃for one minute, extension at 72℃for one minute, total 30 cycles, a final extension at 72℃for ten minutes. It amplified 258-base pair products.4.Direct sequenced the PCR products: The PCR amplification products of five rifampin drug-sensitive strains and twenty rifampin drug-resistance stains and standard control strains were direct sequenced. Results: A 258bp fragments of rpoB gene were amplified from all Mycobacterium tuberculosis including international standard strain and rifampin drug-resistant standard strain, atypical international standard strain and control Streptococcus pneumonias strain was not amplified the fragment. No mutation was found in five rifampin drug-sensitive strains and standard control strains. Mutation of rpoB gene was identified in 19 of 20 rifampin drug-resistant strains, incidence was 95﹪,a total of 8strains had one point mutation at codon position 531 (42﹪), 6 of 8 examples had C→T (Ser→Leu) mutation. A total of 8 strains had one point mutation at codon position 526 (42﹪), 5 of 8 examples had C→T (His→Tyr) mutation. A total of 3 strains had one point mutation at codon position 516. One strain was not found mutation. The correspondence rate of PCR-DS method detection rifampin clinical drug-resistance strains was 95﹪,when compared to the drug sensitivity testing, specificity was 100﹪. This experiment was based on the technique of PCR. PCR technique was an important finding in the molecular biology in the twentieth century, it can obtained a lot of nucleicacid fragment, made people to amplify the specific DNA fragment to millions times fragment. PCR technique had important application value in DNA recombination and expression, gene structure analysis and functional detection. It was especially applied extensively in diagnosis infection disease, can diagnosed specific pathogen, can diagnosed carrier and latent infection, and districted classification and type to pathogen. DNA sequence analysis was another important technique of molecular biology, it can help to explore organization and function of gene, relations between gene and disease, push development of life science research. There are two methods of sequencing, they were enzymatic reaction(dideoxy chain termination) and chemistry lysis. The two methods were artificial operation with disadvantage of complexity, slow speed and radioactivity. Laser sequencing method and automated sequencer was a rapidand accurate automated sequencing method, this method's theory was as same as dideoxy chain termination, laser sequencing used fluorescence stain as labeling, different fluorescence represent different base, detection system put the fluorescence symbol transmit to computer, automated reading sequence by program analysis. Two methods were classified into primer labeling system and fluorescence labeling system. This experiment had used American ABI company 377 model automated laser sequencer to sequence the products of PCR amplification. This experiment identified that key molecular mechanism of rifampin drug-resistance were due to mutations in the rpoB gene of Mycobacterium tuberculosis encoding the β-subunit of the RNA polymerase. Mutations of rpoB gene were identified in 19 of 20 rifampin drug-resistant strains, frequent codon positions were 531 and 526, and mutation type was the point mutation. The correspondence rate of PCR-DS method detection rifampin clinical drug-resistance strains was 95﹪,when compared to the drug sensitivity testing, specificity was 100﹪.It identified that there were high sensitivity and specificity by PCR-DS method. PCR-SSCP method also rapid detected rifampin drug-resistance clinical strains, but did not know the position,characterization,number and frequency of mutation. This experiment method could identify the position,characterization,number and frequency of mutation. This experiment considered that have high significance based on PCR-SSCP. In a word, PCR-DS was a rapid,accurate,simple,cheap method to...
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