[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.


CONCLUSIONS
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.
Shi
Rui-ru
RR
Central Laboratory of Henan Provincial Chest Hospital, Zhengzhou 450003, China. shiruiru@hotmail.com
Du
Chang-mei
CM
D000995:Antitubercular Agents / Q000494:pharmacology*; D024901:Drug Resistance, Multiple, Bacterial / Q000187:drug effects / Q000235:genetics*; D004977:Ethambutol / Q000494:pharmacology*; D005798:Genes, Bacterial; D014644:Genetic Variation; D006801:Humans; D008826:Microbial Sensitivity Tests; D009154:Mutation; D009169:Mycobacterium tuberculosis / Q000187:drug effects / Q000235:genetics* / Q000302:isolation & purification; D015183:Restriction Mapping; D012680:Sensitivity and Specificity
D000995:Antitubercular Agents; D004977:Ethambutol