Spoligotyping of the Mycobacterium tuberculosis complex using on-Chip PCR.

AIMS: The aim of this study was to develop a rapid PCR-based method for spoligotyping of mycobacteria in the microarray format and to compare it to conventional spoligotyping by hybridization. METHODS AND RESULTS: The method employs the On-Chip PCR technique with primers specific for 43 spacers that separate direct repeats (DRs) in the DR region of mycobacterial DNA. The primers were immobilized on gel-based microarrays, and PCR was performed directly on the chips. The PCR fluorescence images were acquired and processed using a portable fluorescence analyzer equipped with dedicated software. Analysis takes 1.5-2 hours and can be carried out on clinical samples without additional handling. The analytical sensitivity of the method was 103 copies of target DNA. The spoligotyping results of 51 samples produced by the proposed method and by conventional reverse hybridization approach were in full concordance. CONCLUSIONS: High throughput capacity, computerized data analysis, compact equipment, and reliable results make the On-Chip PCR an attractive alternative to intra- and interspecific spoligotyping of Mycobacterium tuberculosis complex bacteria.

Spoligotyping of the Mycobacterium tuberculosis complex using on-Chip PCR.

AIMS: The aim of this study was to develop a rapid PCR-based method for spoligotyping of Mycobacteria in the microarray format and to compare it to conventional spoligotyping by hybridization. METHODS AND RESULTS: The method employs the on-Chip PCR technique with primers specific for 43 spacers that separate direct repeats (DRs) in the DR region of mycobacterial DNA. The primers were immobilized on gel-based microarrays, and PCR was performed directly on the chips. The PCR fluorescence images were acquired and processed using a portable fluorescence analyzer equipped with dedicated software. Analysis takes 1.5-2 hours and can be carried out on clinical samples without additional handling. The analytical sensitivity of the method was 103 copies of target DNA. The spoligotyping results of 51 samples produced by the proposed method and by conventional reverse hybridization approach were in full concordance. CONCLUSIONS: High throughput capacity, computerized data analysis, compact equipment, and reliable results make the on-Chip PCR an attractive alternative to intra- and interspecific spoligotyping of Mycobacterium tuberculosis complex bacteria. SIGNIFICANCE AND IMPACT OF STUDY: Fast microarray-based spoligotyping technique using on-Chip PCR was developed.