Detecting KatG,rpoB Gene Mutation In Mycobacterium Tuberculosis By The Reverse Dot-blot Hybridization Method

Recently, tuberculosis(TB) reemergence and spread are of world wide concern due to the rapidly increasing extent of movement of populations, aging, and relying on TB Bacillus Calmette-Guerin the coninfection of HIV.The situation is aggravated by the increasing circulation of multidrug-resistant (MDR) Mycobacterium tuberculosis strains. The standard treatment for TB is a multidrug regimen that is based on isoniazid (INH) and rifampin (RIF), the drugs most efficacious against Mycobacterium tuberculosis infection. The development of resistance to these two drugs, however, means that the efficacy of standard anti-TB treatment is reduced by up to 77%.therefore, The detection of resistant M.tuberculosis strains quickly and accuratly will help to find optimum therapeutic method and prevention for the spread of MDR-TB.INH resistance is apparently controlled by a more complex genetic system that involves several genes,namely, katG, inhA, ahpC, ndh. Between 60%-70% of the INH-resistant strains encode mutations in katG,and a specific mutation (in codon 315 of the katG gene ) is responsible for many of these cases of resistance and the mutations is associated with high -level resistance to isoniazid .in conclusion, we should focus on this mutation and minimize the need to search for other mutations that encode resistance to INH. More than 95% of RIF-resistant strains are associated with mutations within an 69bp core region of the rpoB gene which codon 23 amino acid.the molecular mechanism for RIF resistance is that the mutation in RNA polymerase beta subunit gene which reduced affinity with RFP confer resistance to mycobacterium tuberculosis. So detection of mutations in the 61-core region of rpoB gene is a quite useful strategy for diagnosis.while the frequency of mutation in these hot-spot region is different in every country. An latest article indicate that the codon numbers of the most frequently encountered mutations were 531 (41%), 526 (40%), and 516 (4%)in china.The situation of multidrug-resistant(MDR) mycobacterium tuberculosis in our country is serious.most of which are resistant to frontline drugs INH and RFP. especially to MDR.the resistance of TB have seriously threat the controlling of spreading of TB.so it is important to established advanced methods to detect the resistance of TB,which cancontrol and prevent spreading of MDR.To develop such research, it is quite necessary to detect mutations in katG, rpoB gene accurately and roundly.Particular methods described so far for the detection of katG 315 changes and rpoB gene include single-strand conformation polymorphism(SSCP), RFLP, real-time PCR.These methods have their virtues and disadvantage and most of them can not simultaneously detect resistance to RFP and INH in one reaction, nowdays, commercial detection kit have been used to detect four mutations in codon 526 and 531 (from Innogenetics NV, Belgium) and have characteristic of simple, rapid, specificity and high sensitivity. Howerer, the products can detect only a few mutations and the price is high, mostly important it can not simultaneously detect katG and rpoB in one reaction. The aim of the present study was to established a simple, rapid and sensitive hybridization method for detecting drug resistance relevant gene mutation in Mycobacterium tuberculosis.that can simultaneously detect resistance to RIF and INH in one nylon membranes.Reverse dot-blot hybridization method based on PCR and reverse hybridization technology can rapidly detect DNA mutation in samples, the sensitivity and specificity of which is associated with probe design,hybridization temperature and efficiency of PCR amplification our study successfully solve the inhomogeneity and not-specificity problem encounted in hybridization where there are many probes and design a new genotypic approach that simultaneously detect resistance to RIF and INH in one nylon membranes.1. The distribution of 315 mutation in INH resistance in china and Detecting katG gene mutation in mycobacterium tuberculosis by the reverse dot-blot hybridization method Thirty INH resistance mycobacterium tuberculosis isolates were recovered from Anhui province center for disease control and prevention(AHCDC) (INH MICs: 0.5ug/ml).DNA sequencing was performed for kagG of the INHr strains.Amplicons were obtained and sent to huanuo company.of thirty INHr isolates,eighteen isolates were found mutations in codon 315 among which The mutation AGC—>ACC at condon 315 in katG was found in 53%(16 of 30)of the resistant strains ,the other isolates were rare mutations(AGC—>AAC)which have been reported abroad and areassociated with high-level resistance to isoniazid.we did not find AGC—>ACA mutation probably because of a small quantity of isolates.the experiment showed that the occurrence of mutations in 315 of katG was high in INH resistant strains and we should focus on this mutation.In order to establish reverse dot-blot hybridization method.we selected one sensitive strain and one resistance strain,cloning PCR product of katG with these two strains into pUCm-T vector.four single-strand specific probes designed to detect mutated and wild katG gene in mycobacterium tuberculosis were spotted and fixed on nylon membranes under 80°C.PCR products labeled with biotin were obtained by using downstream primer labeled with biotin ,then hybridized and analyzed with streptavidin HRP and TMB.when the PCR products amplified by using R Plasmid hybridize with nylon membrane in EP tube ,only R probe show hybridization spot while S probe have faintness spot.In order to show the following opinion: compare to hybridization in Hybridization oven .Hybridization in EP tube can reduced non-specificity hybridization sharply which had good sensitivity and not-specificity problem was under control.the hybridization was done by using R plasmid amplification products with nylon membranes in two different conditions(in hybridization oven and EP tube)the two hybridization temperature and hybridization process were the same .from the pictures got in hybridization we can see only R probe have spot while the other three probes were blank when the hybridization was done in EP tube,while hybridization was done in hybridization oven.there have non-specificity hybridization spot in S probe.these result showed compare to hybridization in Hybridization oven the result can reduced non-specificity hybridization sharply in Eppendorf tube According to the DNA sequence near to 315 condon ,we design a 19bp probe that the mismatch base was 12bp to 5'terminal and 6bp to 3'terminal.this design can sove non- specificity successfully.2. Detecting katG gene and rpoB gene mutation in mycobacterium tuberculosis by the reverse dot-blot hybridization method. In order to detect resistance to RIF and INH in one nylon membranes we design two...