"Spoligoriftyping," a dual-priming-oligonucleotide-based direct-hybridization assay for tuberculosis control with a multianalyte microbead-based hybridization system.
We developed "spoligoriftyping," a 53-plex assay based on two preexisting methods, the spoligotyping and "rifoligotyping" assays, by combining them into a single assay. Spoligoriftyping allows simultaneous spoligotyping (i.e., clustered regularly interspaced short palindromic repeat [CRISPR]-based genotyping) and characterization of the main rifampin drug resistance mutations on the rpoB hot spot region in a few hours. This test partly uses the dual-priming-oligonucleotide (DPO) principle, which allows simultaneous efficient amplifications of rpoB and the CRISPR locus in the same sample. We tested this method on a set of 114 previously phenotypically and genotypically characterized multidrug-resistant (MDR) Mycobacterium tuberculosis or drug-susceptible M. tuberculosis DNA extracted from clinical isolates obtained from patients from Bulgaria, Nigeria, and Germany. We showed that our method is 100% concordant with rpoB sequencing results and 99.95% (3,911/3,913 spoligotype data points) correlated with classical spoligotyping results. The sensitivity and specificity of our assay were 99 and 100%, respectively, compared to those of phenotypic drug susceptibility testing. Such assays pave the way to the implementation of locally and specifically adapted methods of performing in a single tube both drug resistance mutation detection and genotyping in a few hours.
Institut de Génétique et Microbiologie, CNRS-Université Paris-Sud, IGEPE Team, Paris, France.
Michel Kiréopori
MK
Gomgnimbou
Edgar
E
Abadia
Jian
J
Zhang
Guislaine
G
Refrégier
Stefan
S
Panaiotov
Elizabeta
E
Bachiyska
Christophe
C
Sola
D000995:Antitubercular Agents / Q000494:pharmacology; D002031:Bulgaria; D012321:DNA-Directed RNA Polymerases / Q000235:genetics; D024881:Drug Resistance, Bacterial; D005838:Genotype; D005858:Germany; D006801:Humans; D008826:Microbial Sensitivity Tests / Q000379:methods; D008863:Microspheres; D058889:Molecular Typing / Q000379:methods*; D009169:Mycobacterium tuberculosis / Q000145:classification* / Q000187:drug effects / Q000235:genetics*; D009549:Nigeria; D009693:Nucleic Acid Hybridization / Q000379:methods*; D009841:Oligonucleotides / Q000235:genetics; D012293:Rifampin / Q000494:pharmacology; D018088:Tuberculosis, Multidrug-Resistant / Q000175:diagnosis / Q000382:microbiology*
D000995:Antitubercular Agents; D009841:Oligonucleotides; D012321:DNA-Directed RNA Polymerases; C105078:RNA polymerase beta subunit; D012293:Rifampin