Single-tube protocol for culture-independent spoligotyping of Mycobacterium tuberculosis based on MeltArray

Abstract

ABSTRACT 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. IMPORTANCE 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. 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 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 and thus sufficient for analyzing both culture and sputum samples. We conclude that MeltArray-based spoligotyping could be used immediately in low- and middle-income countries with a high tuberculosis burden, given its easy access, improved throughput, and potential applicability to clinical samples.