| In recent years, epidemiological situation of tuberculosis is presenting the tendency ofraise. One of the main causes is that the disease caused by the drug-resistance ofMycobacterium tuberculosis, especially the multidrug-resistance MTB is prevailing. Thesituation of drug resistance for TB in our country is much worse. According to the statistics,the total rate of drug-resistance is 27.8％, and the multi drug-resistance rate reaches as high as10.7％. Because of this the tuberculosis prevalence rate and the mortality rate stay at a highlevel. So at the present it is the emergency to create the rapid and precise methods for the drugresistance diagnosis on the national plan for the tuberculosis preventation and control and itsthe goal realization, and the important base of making efficient chemotherapy to the disease.Isoniazid (INH) is one of the most potent antituberculosis drugs. The mechanism ofMTB resistance to INH is clear primarily along with the development of molecularbiotechnological methods. It is verified that INH-resistant MTB strains have a mutationprincipally in katG, pre-inhA, inhA, ndh and oxyR-ahpC genes.To investigate situation of the gene mutations in MTB and identify the characteristic andthe distribution of the mutation,clinical isolates of MTB from the patients with tuberculosis in7 provinces including Xizang, Hunan, Henan, Sichuan, Fujian, Anhui, and Shanxi in Chinawere collected and identified by means of biochemistry reaction method.Thedrug-susceptibility of the isolates was determinated by proportional drug susceptibility testing.Then the fragment of katG, pre-inhA, inhA, ndh and oxyR-ahpC genes was amplified by PCRand the mutations in the gene fragments were detected by DNA sequencing. Based on thesequencing result, the mutation was analyzed, including the rate of every mutation site, thekinds of the gene mutation in MTB and the correlation between the gene mutation anddrug-resistance.536 clinical isolates of Mcobacterium from the patients with tuberculosis were identifiedby 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 non-mycobacterium tuberculosis (NTM) by which the patient wasco-infected. The drug-susceptibility of 482 clinical isolates of MTB were determinated by theproportional drug susceptibility testing. The results showed that 126 strains (26.14％) weresensitive to all 5 drugs, including INH, Rifarmpicin (RFP), Streptomycin (SM), Ethambutol(EMB), and Para-aminosalicylic acid (PAS). The results showed 118(24.48％) for sensitivecompletely, 364 (75.52％) for drug-resistant in which there were 88 (18.26％) for the singleand 276 (57.26％) for the multi-resistance.It was designed a pairs of primer for the katG gene, the pre-inhA gene, the inhA gene, thendh gene and the oxyR-ahpC gene respectively. Then the polymerase chain reaction (PCR)and the DNA sequence analysis were carried on to detect the gene mutations in MTB standardstrain H37Rv and 291 MTB clinical isolates from which the background material of the paitentwith tuberculosis was clear, including 64 strains were sensitive absolutely to INH, 48sensitive relatively to INH, 30 single drug-resistance to INH only, 149 multi drug-resistantce.The results of DNA sequencing showed: (1) In 179 INH-resistance strains, 163 strainshad been found the gene mutation in the "hot spot mutation area" and in 112 INH-sensitiveatrains, 3 strains had been found KatG gene mutation. Compared with the proportional drugsusceptibility testing, the sensitivity and specificity of DNA sequencing were 91.06％(163/179) and 97.32％(109/112) respectively, the positive forecast value and the negativeforecast value were 98.19％(163/166) and 87.20％(1.09/125) respectively. The concordancerate between the proportional drug susceptibility testing and DNA sequencing was 93.47％(272/291). There was no significant difference between the two methods by Chi-squarestatistics test (x2=7.58, P＞0.05). (2) In 179 INH-resistance strains, 129 strains (72.09％) hadbeen found the gene mutation at 463 spot, and in 112 drug-sensitive, 74 strains (66.07％) hadbeen found gene mutation at the same spot. There was no significant difference by Chi-squarestatistics test (x2=1.1739, P＞0.05). Therefore, it is confirmed that the gene mutation at 463spot has no correlation with INH-resistance. (3) In the 179 INH-resistance strains there were138 (77.09％) which had gene mutation in KatG, and of which 7 positions has not seen anyreport now;131(73.18％) happened mutation in the position of 315, involveing 4 kinds ofmutation types, but the main mutation locus was AGC (Ser)->ACC (Thr), which happened in123 strains (93.89％). But we have not found delation or insertation for KatG gene in any INH-resistance strains. (4) There was 1 strain (0.56％) in INH-resistance strains for inhAwhile in the sensitive strains was not.(5) 18 (10.06％) INH-resistance strains have genemutation in oxyR-ahpC gene connection, in which 7 strains (3.39％) have the -9C -> Tmutation, 3 strains have the -5G -> A mutation, 3 strains have the -14C -> T change and so on;while in the sensitive strains was not any change. (6) 3 (1.68％) INH-resistance strains havegene mutation in ndh and the type is Glu191Gly, while in the sensitive strains was not anychange. (7) In 179 INH-resistance strains, There was no mutation in INH-sensitive aboutpre-inhA gene. Among 179 clinical isolates,there were 12 strains had the change of the -15C->T, and in which 9 strains happened single locus mutation but does not have other spots andother related gene mutations. The other mutations, such as -19G -> A, -8T -> C and 13G -> C,had also been seen in.some stains. While in the sensitive strains was not any change. (8)There was no oxyR and ahpC gene mutation in all of the Strains. (9) Synthesizing variousgenes changes in this experiment, we conclude that there are 163 strains to occur with theINH- resistance correlation the gene mutations.This research further confirmed the relationship between Mycobacterium tuberculosisINH resistance and katG, pre-inhA, inhA, ndh, oxyR-ahpC gene mutations.At the same time,it provides the foundation for the probe design for the next step, simultaneously alsoprompted that there is also other mechanisms to participate in the INH-resistance.
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