RESUMEN
Objective To study and preliminarily evaluate the standard spacer oligonucleotide typing (Spoligotyping) method and the application on Mycobacterium tuberculosis (M.tuberculosis).Methods Spoligotyping on 224 M.tuberculosis strains was studied by the molecular biological techniques, including DNA isolation, PCR, reverse line blot hybridization, together with data analysis software BioNumerics (Version 5.0). Results Standardization on both Spoligotyping method and parameters in result analysis and the usage of the analysis software were studied. Through this method, 224 M.tuberculosis clinical strains were classified into 2 clusters including 129 Beijing family strains and 95 non-Beijing family strains. The predominant strains belonged to Beijing family. Conclusion Standard Spoligotyping method was preliminary determined in China, showing that it was a simple, rapid, and robust method for simultaneous detection and typing of M.tubereulosis. This method can be used for tracing the source of infection and understanding the epidemic trend of M.tuberculosis. Spoligotyping can also be served as a method for simultaneous detection and typing ofM.tuberculosis, and to identify Beijing family strains.
RESUMEN
Objective To establish and evaluate the standardized protocol of multiple loci variable numbers tandem repeats (VNTR) analysis (MLVA) for genotyping Mycobacterium tuberculosis (M.tuberculosis).Methods 15 VNTR loci were chosen for genotyping 54 Chinese M.tuberculosis strains by PCR-electmphoresis-based VNTR analysis and the results were analyzed by software BioNumerics (Version 5.0).Results MLVA method was successfully established and standardized,including the standard protocol for bacterial culture,DNA isolation,PCR and agarose gel electrophoresis and the software analysis. 15 VNTR loci were confirmed,including ETRA,ETRB,ETRC,ETRD,ETRE,MIRU10,MIRU16,MIRU23,MIRU26,MIRU27,MIRU39,MIRU40,Mtub21,Mtub30 and Mtub39,to be suitable for MLVA analysis of M.tuberculosis.Conclusion The standardized MLVA method has been established successfully.This method is simple and has powerful capacity for genotyping and strain differentiation,can be used for the network surveillance on pathogens of M.tuberculosis,and the data are comparable between laboratories.It is valuable for tracing the source and studying the trend of prevalence during investigation of M.tuberculosis infections.