Mycobacterium tuberculosis complex CRISPR genotyping: improving efficiency, throughput and discriminative power of 'spoligotyping' with new spacers and a microbead-based hybridization assay.
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.
Zhang
Jian
J
IGEPE Team, Institute of Genetics and Microbiology, UMR8621, Universud, CNRS Université Paris-Sud 11, Campus d'Orsay, F-91405 Orsay-Cedex, France.
Abadia
Edgar
E
IGEPE Team, Institute of Genetics and Microbiology, UMR8621, Universud, CNRS Université Paris-Sud 11, Campus d'Orsay, F-91405 Orsay-Cedex, France.
Refregier
Guislaine
G
IGEPE Team, Institute of Genetics and Microbiology, UMR8621, Universud, CNRS Université Paris-Sud 11, Campus d'Orsay, F-91405 Orsay-Cedex, France.
Tafaj
Silva
S
National TB Reference Laboratory, University Hospital of Lung Diseases 'Shefqet Ndroqi', Tirana, Albania.
Boschiroli
Maria Laura
ML
Agence Française de Sécurité Sanitaire des Aliments, Maisons-Alfort, France.
Guillard
Bertrand
B
Institut Pasteur du Cambodge, Pnom-Penh, Cambodia.
Andremont
Antoine
A
Microbiology Laboratory, Bichat-Claude Bernard Hospital, Paris, France.
Ruimy
Raymond
R
Microbiology Laboratory, Bichat-Claude Bernard Hospital, Paris, France.
Sola
Christophe
C
Unité de Génétique Mycobactérienne, Institut Pasteur, Paris, France.|IGEPE Team, Institute of Genetics and Microbiology, UMR8621, Universud, CNRS Université Paris-Sud 11, Campus d'Orsay, F-91405 Orsay-Cedex, France.
D015373:Bacterial Typing Techniques / Q000191:economics / Q000379:methods*; D016172:DNA Fingerprinting / Q000191:economics / Q000379:methods*; D005838:Genotype; D006801:Humans; D008863:Microspheres; D009169:Mycobacterium tuberculosis / Q000145:classification* / Q000235:genetics*; D009693:Nucleic Acid Hybridization / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D014376:Tuberculosis / Q000382:microbiology*