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Spoligotyping of the Mycobacterium tuberculosis complex using on-Chip PCR.
Sergey Lapa1, Alexey Kuzmin2, Larisa Сhernousova2
1Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow 119991, Russia.
Journal of Applied Microbiology
|January 10, 2023
Summary
A new microarray-based method using on-chip PCR rapidly performs spoligotyping for Mycobacteria. This technique offers a fast, reliable alternative for analyzing Mycobacterium tuberculosis complex bacteria.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- Spoligotyping is crucial for Mycobacterium species identification.
- Conventional spoligotyping methods can be time-consuming and labor-intensive.
Purpose of the Study:
- To develop a rapid, microarray-based PCR method for Mycobacteria spoligotyping.
- To compare the novel method's performance against conventional spoligotyping.
Main Methods:
- On-Chip PCR technique utilizing primers for 43 spacers in the DR region of mycobacterial DNA.
- Immobilization of primers on gel-based microarrays with direct PCR performed on-chip.
- Analysis of PCR fluorescence images using a portable analyzer with dedicated software.
Main Results:
- The developed method provides spoligotyping results in 1.5-2 hours.
- Analytical sensitivity was determined to be 10^3 copies of target DNA.
- Full concordance was observed between the on-Chip PCR method and conventional reverse hybridization for 51 samples.
Conclusions:
- On-Chip PCR offers a high-throughput, computerized, and reliable alternative for spoligotyping.
- The developed microarray-based technique is suitable for both intra- and interspecific spoligotyping of Mycobacterium tuberculosis complex bacteria.
