McSpoligotyping, a One-Step Melting Curve Analysis-Based Protocol for Spoligotyping of Mycobacterium tuberculosis

The direct repeat (DR) region in the Mycobacterium tuberculosis (MTB) genome is composed of highly polymorphic direct variant repeats, which are the basis of spacer oligonucleotide typing (spoligotyping) to study the population structure and epidemiology of M. tuberculosis However, the membrane hybridization-based detection format requires various post-PCR manipulations and is prone to carryover contamination, restricting its wide use in high-TB-burden and resource-limited countries. We developed a one-step spoligotyping protocol, termed McSpoligotyping, based on real-time PCR. The typing results can be generated within 3 h by a single step of DNA addition. When evaluated with a collection of 1,968 isolates of MTB, McSpoligotyping agreed 97.71% (1,923/1,968) by sample and 99.93% (84,568/84,624) by spacer with traditional spoligotyping. Sequencing results showed that McSpoligotyping was even more accurate than spoligotyping (99.34% versus 98.37%). Further exploration of the false results of McSpoligotyping revealed the presence of single-nucleotide polymorphisms in the DR region. We concluded that McSpoligotyping could be used in epidemiology studies of tuberculosis by taking advantage of the shortened procedure, ease of use, and compatibility of results with standard spoligotyping.