Simple multiplex PCR assay for identification of Beijing family Mycobacterium tuberculosis isolates with a lineage-specific mutation in Rv0679c

Abstract

The Beijing genotype of Mycobacterium tuberculosis is known to be a worldwide epidemic clade. It is suggested to be a possibly resistant clone against BCG vaccination and is also suggested to be highly pathogenic and prone to becoming drug resistant. Thus, monitoring the prevalence of this lineage seems to be important for the proper control of tuberculosis. The Rv0679c protein of M. tuberculosis has been predicted to be one of the outer membrane proteins and is suggested to contribute to host cell invasion. Here, we conducted a sequence analysis of the Rv0679c gene using clinical isolates and found that a single nucleotide polymorphism, C to G at position 426, can be observed only in the isolates that are identified as members of the Beijing genotype family. Here, we developed a simple multiplex PCR assay to detect this point mutation and applied it to 619 clinical isolates. The method successfully distinguished Beijing lineage clones from non-Beijing strains with 100% accuracy. This simple, quick, and cost-effective multiplex PCR assay can be used for a survey or for monitoring the prevalence of Beijing genotype M. tuberculosis strains.

Fig 1

PCR primers and products of Rv0679c-targeting multiplex PCR for Beijing lineage discrimination. (A) In the Beijing sample, the 163-bp product is amplified more dominantly than is the 261-bp product. (B) In the non-Beijing sample, 163-bp product is not amplified because of the mismatch of the 3′ end of R1. Fw, forward primer; R1, reverse primer 1 (Beijing lineage specific); R2, reverse primer 2. Two-base noncomplement nucleotides at the 5′ end are shown by black squares.

Fig 2

Electrophoresis results of the multiplex PCR products. Lane M, 50-bp ladder DNA size marker; lane 1, M. bovis BCG Tokyo 172 (non-Beijing lineage control) strain; lane 2, M. tuberculosis OM-9 strain (Beijing lineage control); lane 3, M. tuberculosis H37Rv; lane 4, M. africanum ATCC 25420; lanes 5–8, M. tuberculosis clinical isolates (lane 5, non-Beijing east Asian; lane 6, EAI; lane 7, LAM9; lane 8, Beijing); lane 9, M. avium strain JATA51-1; lane 10, M. kansasii JATA21-1; lane NC, negative control.