Mycobacterium tuberculosis complex CRISPR genotyping: improving efficiency, throughput and discriminative power of ‘spoligotyping’ with new spacers and a microbead-based hybridization assay

• DOI: 10.1099/jmm.0.016949-0
• Publication Date: 2009-12-04T03:28:10Z
• Authors: Jian Zhang; Edgar Abadia; Guislaine Refregier; Silva Tafaj; Maria Laura Bosch...; Bertrand Guillard; Antoine Andremont; Raymond Ruimy; Christophe Sola
• Keywords: MESH: Mycobacterium tuberculosis; Genotype; MESH: Microspheres; Sensitivity and Specificity; MESH: Bacterial Typing Techniques; MESH: Genotype; MESH: Nucleic Acid Hybridization; 03 medical and health sciences; MESH: DNA Fingerprinting; Humans; Tuberculosis; MESH: Tuberculosis; [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology; 0303 health sciences; MESH: Humans; Nucleic Acid Hybridization; Reproducibility of Results; Mycobacterium tuberculosis; DNA Fingerprinting; MESH: Sensitivity and Specificity; Microspheres; Bacterial Typing Techniques; 3. Good health; MESH: Reproducibility of Results; [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology

The aims of the present study were to implement a microbead-based ‘spoligotyping’ technique and to evaluate improvements by the addition of a panel of 25 extra spacers that we expected to provide an increased resolution on principal genetic group 1 (PGG 1) strains. We confirmed the high sensitivity and reproducibility of the classical technique using the 43 spacer panel and we obtained perfect agreement between the membrane-based and the microbead-based techniques. We further demonstrated an increase in the discriminative power of an extended 68 spacer format for differentiation of PGG 1 clinical isolates, in particular for the East African–Indian clade. Finally, we define a limited yet highly informative reduced 10 spacer panel set which could offer a more cost-effective option for implementation in resource-limited countries and that could decrease the need for additional VNTR (variable number of tandem repeats) genotyping work in molecular epidemiological studies. We also present an economic analysis comparing membrane-based and microbead-based techniques.