Rapid Detection of Resistance Associated Mutations in Mycobacterium tuberculosis by LightCycler PCR

Molecular Characterization of Rifampin- and Isoniazid-Resistant Mycobacterium tuberculosis Strains Isolated in Poland

“…For the detection of mutations in the inhA locus, a pair of conventional FRET probes was used (anchor-sensor design) (26). The 5Ј-Red 640-labeled sensor (TB ATT) covered the segment containing the regulatory region ( Table 1).…”

“…Twenty-six Inh r M. tuberculosis strains that had no mutations at katG codon 315 (including one strain carrying a mutation at codon 279) were investigated in a real-time PCR test. A set of standard FRET probes was used to detect changes in a 22-bp fragment upstream of the start site of inhA (26). For 4 INHsusceptible control isolates and 14 Inh r strains (including one strain with a mutation at codon 279 of katG), the T m s were within the range obtained for the reference strain, and therefore they were not considered mutants (Table 3).…”

“…Since almost 15% of the strains studied had a C3T nucleotide substitution at position 15 upstream of the start site of the inhA gene, we applied a pair of standard FRET probes to search for mutations in the regulatory region of inhA (26). Mutations in this region cause the overexpression of this gene.…”

Direct Detection of Rifampin- and Isoniazid-Resistant Mycobacterium tuberculosis in Auramine-Rhodamine-Positive Sputum Specimens by Real-Time PCR

“…All DNA extracts were amplified with the primers used in conventional PCR, with the addition of the fluorescein and Red 640-labeled probes to the amplification mix. Four different pairs of the probes were used, rpo1 anchor-sensor that covered the region containing codons 526 and 531 of the rpoB gene (22), rpo2 anchor-sensor that covered the region containing codons 510 to 528 of the rpoB gene (23), kat1 anchor-sensor that covered the region containing codon 315 of the katG gene (22), and inh1 anchor-sensor that detected nucleotide substitution C209T in the regulatory region of the inhA gene (23). All primers and hybridization probes were synthesized by TIB MOLBIOL (DNA Synthesis Service; Roche Diagnostics, Berlin, Germany).…”

Computational prediction of the melting temperature of a DNA biosensor to detect Mycobacterium tuberculosis

“…Various molecular methods have been developed to rapidly detect M. tuberculosis strain in the insertion sequence 6110 (IS6110) region, IS6110 is an insertion element found exclusively within the members of the M. tuberculosis complex and, because of this exclusivity, it has become an important diagnostic tool in the identification of M. tuberculosis complex species. Some methods including the line probe assay [2], singlet-strand conformational polymorphism (SSCP), Polymerase Chain Reaction (PCR) [3,4], and real-time PCR [5][6][7]. The purpose of a PCR is to make a huge number of copies of a gene.…”