RESUMEN
OBJECTIVE: To determine the rpsL and rrs gene mutation in Mycobacterium tuberculosis (M. tuberculosis) and compare the consistency between the results of denaturing high-performance liquid chromatography (DHPLC) and those of DNA sequencing. METHODS: The values of streptomycin minimum inhibitory concentration (MIC) against 215 M. tuberculosis clinical isolates, 115 being streptomycin-resistant and 100 being susceptible by a routine proportional method, were tested by DHPLC. DNA sequencing was conducted to detect the rpsL and rrs mutation. RESULTS: 98 of the 115 streptomycin-resistant isolates (85.2%) harbored rpsL and/or rrs mutation, 76.5% of which being rpsL mutation (88/115). There was no significant correlation between the MIC values and mutation types. No mutation was found in all the susceptible isolates. There was a complete consistency between the DHPLC results and those of DNA sequencing. CONCLUSION: DHPLC can be regarded as a useful and powerful tool to detect the streptomycin resistance detection in M. tuberculosis.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Farmacorresistencia Bacteriana , Mycobacterium tuberculosis/efectos de los fármacos , Estreptomicina/farmacología , Adolescente , Adulto , Anciano , Antibacterianos/farmacología , Proteínas Bacterianas/genética , Análisis Mutacional de ADN , ADN Bacteriano/química , ADN Bacteriano/genética , Humanos , Pruebas de Sensibilidad Microbiana , Persona de Mediana Edad , Mutación , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/aislamiento & purificación , Reproducibilidad de los Resultados , Proteínas Ribosómicas/genética , Tuberculosis/microbiología , Adulto JovenRESUMEN
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.