Simple and rapid discrimination of embB codon 306 mutations in Mycobacterium tuberculosis clinical isolates by a real-time PCR assay using an LNA-TaqMan probe

Single nucleotide polymorphisms in the codon 306 of embB gene are most frequently reported in ethambutol-resistant Mycobacterium tuberculosis clinical isolates. Here, we report a simple and rapid real-time PCR assay using a locked nucleic acid (LNA)-TaqMan probe for discriminating the embB306 mutations. The use of a 15-bp chimeric LNA/DNA probe led to a relatively higher level of sensitivity and fluorescence signal in the wild-type embB306 ATG codon. Therefore, the mutant alleles were easily distinguishable from the wild-type allele by their distinctive amplification curve shapes without a melting analysis of the PCR product. This system was fast and less than 0.1pg of genomic DNA per reaction was needed for detection. Because the results from this real-time assay were absolutely consistent with those from DNA sequencing, it can be effectively applied as a simple and rapid method for primary screening of embB306 mutations that occur frequently in ethambutol-resistant and/or multidrug-resistant M. tuberculosis isolates. ► We develop a rapid and simple real-time PCR assay for discriminating embB306 SNPs. ► This assay uses a LNA-TaqMan probe instead of a normal DNA-TaqMan probe. ► This system requires only less than 0.1pg of genomic DNA per reaction. ► Mutant is easily distinguishable by its amplification curve without melting analysis.