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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. [ABSTRACT FROM AUTHOR]

Aptamers are nucleic acid-based scaffolds that can bind with high affinity to a variety of biological targets. Aptamers are identified from large DNA or RNA libraries through a process of directed molecular evolution (SELEX). Chemical modification of nucleic acids considerably increases the functional and structural diversity of aptamer libraries and substantially increases the affinity of the aptamers. Additionally, modified aptamers exhibit much greater resistance to biodegradation. The evolutionary selection of modified aptamers is conditioned by the possibility of the enzymatic synthesis and replication of non-natural nucleic acids. Wild-type or mutant polymerases and their non-natural nucleotide substrates that can support SELEX are highlighted in the present review. A focus is made on the efforts to find the most suitable type of nucleotide modifications and the engineering of new polymerases. Post-SELEX modification as a complementary method will be briefly considered as well. [ABSTRACT FROM AUTHOR]

Mycobacterium tuberculosis remains a leading cause of morbidity and mortality worldwide. This circumstance underscores the relevance of population studies of tuberculosis for transmission dynamics control. In this study, we describe a conversion of the spoligotyping of M . tuberculosis complex isolates on a platform of custom designed hydrogel microarrays (biochips). An algorithm of automated data processing and interpretation of hybridization results using online database was proposed. In total, the 445 samples were tested. Initially, 97 samples representing multiple species of M . tuberculosis complex and nontuberculous mycobacteria were used for protocol optimization and cut-off settings. The developed assay was further evaluated on the out-group of the 348 mycobacterial samples. Results showed high concordance with the conventional membrane-based spoligotyping method. Diagnostic sensitivity and diagnostic specificity of the spoligo–biochip assay were 99.1% and 100%, respectively. The analytical sensitivity was determined to be 500 genomic equivalents of mycobacterial DNA. The high sensitivity and specificity, ease of operation procedures, and the automatic processing of measured data make the developed assay a useful tool for the rapid and accurate genotyping of M. tuberculosis . [ABSTRACT FROM AUTHOR]