Single-tube Protocol for Culture-Independent Spoligotyping of Mycobacterium Tuberculosis Based on MeltArray.

Spacer oligonucleotide typing (spoligotyping), the first-line genotyping assay for Mycobacterium tuberculosis (MTB), plays a fundamental role in the investigation of its epidemiology and evolution. However, the traditional spoligotyping protocol was established by using the reverse dot blot hybridization technique, which is characterized by a complex procedure, long turnaround time, and subjectivity in result interpretation, thus hindering its widespread use in low- and middle-income countries (LMICs) with a high tuberculosis burden. In this study, we established a single-tube spoligotyping assay using MeltArray, a highly multiplex polymerase chain reaction (PCR) approach that runs on a real-time PCR thermocycler. The MeltArray protocol included an internal positive control, gyrB, to indicate the abundance of MTB via the quantification cycle (Cq) and 43 spacers to identify the spoligotype via melting curve analysis. The entire protocol was completed in a single step within 2.5 hours. The lowest detectable copy number for the tested strains was 20 copies/reaction. In a blind evaluation of 318 MTB isolates, the MeltArray assay yielded 98.1% (312/318) concordance with the traditional approach and 100.0% with Sanger sequencing. Further evaluation of 151 liquid culture-matched sputum samples showed that all qualified (Cq <35) sputum samples (80.8%, 122/151) yielded results consistent with those of the liquid culture samples, including 97.5% (119/122) single spoligotypes and 2.5% (3/122) mixed spoligotypes. We conclude that MeltArray-based spoligotyping could be used immediately in LMICs, given its easy access, improved throughput, and potential applicability to clinical samples.