“Spoligoriftyping,” a Dual-Priming-Oligonucleotide-Based Direct-Hybridization Assay for Tuberculosis Control with a Multianalyte Microbead-Based Hybridization System

Abstract

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.

Tuberculosis-Spoligo-Rifampin-Isoniazid Typing: an All-in-One Assay Technique for Surveillance and Control of Multidrug-Resistant Tuberculosis on Luminex Devices

“…We designed two pairs of dual-priming oligonucleotide (DPO) primers to simultaneously amplify 180-bp fragments of the katG gene (overlapping the codon 315 position) and the inhA promoter (overlapping the Ϫ15 and Ϫ8 positions) within the multiplex PCR previously described (22). All the primers used are shown in Table 1; they include probes/primers previously described for high-throughput spoligotyping (27), spoligoriftyping (22), and the RIOT method (17), as well as in this study. In total, 43 probes were used for spoligotyping, 10 probes for rpoB hot-spot direct and indirect mutation detection, and 6 probes for the katG and inhA genes (katG codon 315 and InhA positions Ϫ15 and Ϫ8) (17,22,28).…”

“…The PCRs and PCR product hybridization have been described previously (22). The protocol for the MagPix is the same as that for the Luminex 200 except that the use of magnetic beads is mandatory.…”

“…We previously reported on the development of spoligoriftyping, a method that combines spoligotyping and rifoligotyping (22)(23)(24). We now extend the method to the detection of INH resistance-associated mutations within katG and inhA genes.…”

Validation of a Microbead-Based Format for Spoligotyping of Legionella pneumophila

“…The principle of interpretation of the LP-SPOL (L. pneumophila spoligotyping) raw values obtained on Luminex 200 or on Magpix is the same as the one used previously for other microbead-based methods (17). The positive and negative cutoffs used to interpret whether a spacer is positive or negative were computed using the raw results output file.…”

The Role of CRISPR-Cas Systems in Virulence of Pathogenic Bacteria

“…Initially, before an understanding of their physiological role, CRISPR-Cas systems were found to be a useful tool for typing bacterial diversity (23). In Mycobacterium tuberculosis (type III-A), for instance, CRISPR variability has been a gold standard for routine genotyping purposes and to study the epidemiology of M. tuberculosis (24)(25)(26)(27). However, limitations exist for the use of CRISPR typing in evolutionary studies, because it is impossible to study which or how many evolutionary events are responsible for the loss of a cluster of neighboring spacers from the CRISPR (27).…”