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

Objective: Surveyor nuclease is a member of the family of plant endonucleases that cleave heteroduplexes 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 ATG-->GTG, 3 with ATG-->ATT, 5 with ATG-->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.