特异性核酸内切酶检测结核分枝杆菌的乙胺丁醇耐药基因突变

Analysis of ethambutol drug-resistance gene mutation in Mycobacterium tuberculosis by specific endonuclease

目的探讨 Surveyor 酶法在结核分枝杆菌耐药基因突变中的应用。方法采用Surveyor 酶法检测结核分枝杆菌临床分离株60株,其中已经测序证实有 embB 基因突变的乙胺丁醇耐药株45株,无基因突变的敏感株15株。经聚合酶链反应扩增,杂交形成异源双链,Surveyor 酶切,通过聚丙烯酰胺凝胶电泳判断其有无突变存在。结果 15株敏感株均无 embB 基因突变,45株耐药株均存在 embB 基因突变热点306位密码子的点突变,其中33株为 ATG→GTG,3株为 ATG→ATT,5株为 ATG→ATA,2株为 ATG→ATC,2株为 ATG→CTG。经 Surveyor 酶法测定,45株 embB 基因突变株均呈现2条带形,15株敏感株均显示1条带形。结论 Surveyor 酶法与测序法的结果完全相符。采用 Sunreyor 酶法进行异源双链分析,具有简便、稳定、低耗、灵敏性和特异性高的特点。

Objective Surveyor nuclease is a member of the family of plant endonucleases that cleave heteroduplexed DNA with high specificity at sites of base substitution mismatch and DNA distortion. Heteroduplex analysis by Surveyor nuclease is a relatively new method. The purpose of this study was to explore the application of this method in analysis of drug-resistance gene mutation in Mycobacterium tuberculosis. Methods Surveyor nuclease was used to analyze embB gene mutation of 60 Mycobacterium tuberculosis isolates, among which 45 were EMB-resistant and embB gene mutation was found by sequencing, and 15 EMB-susceptible isolates without embB mutation. PCR amplification was carried out first, and then hybridized with products of H37Rv to form heteroduplex, cleaved by Surveyor nuclease, and lastly the results were shown by polyacrylamide gel electrophoresis and mutation was judged according to electrophoresis profile. Results Forty-five EMB-resistant isolates were found harboring embB gene mutation, while in the 15 EMB-susceptible isolates no mutation was found. All the 45 EMB-resistant isolates were revealed to harbor embB gene hotspot codon 306 mutation, among which 33 with ATC→GTG,3 with ATC→ATY,5 with ATC→ATA, 2 with ATG→ATC, 2 with ATG→ CTG. These results from Surveyor nuclease method was completely parallel to those of sequencing. Conclusion Heteroduplex analysis by Surveyor nuclease may become a useful method for drug-resistance gene mutation analysis for Mycobacterium tuberculosis because of its simplicity, stability, low-cost, and high sensitivity and specificity.