A Pyrosequencing assay for rapid recognition of SNPs in Mycobacterium tuberculosis embB306 region.
Ethambutol (EMB) is in use worldwide as a first-line anti-tuberculosis drug and substitutions in codon 306 of the embB gene are the most common mutations found in EMB resistant Mycobacterium tuberculosis (MTB) strains. Pyrosequencing is a real time sequencing method able to rapidly detect mutations in a large number of samples. Using this technique we analyzed, in parallel with conventional sequencing, a 24 bp region of the embB gene of 28 MTB clinical isolates. Pyrosequencing efficiently identified all embB306 mutations, detecting three different single-base substitutions leading to 2 amino acid changes (Met to Val or Ile). Mutated embB alleles were detected in 2 multidrug-resistant (MDR) EMB-susceptible strains. Overall, our results demonstrated that the Pyrosequencing method efficiently recognizes mutations in embB in a very short time and represents a valid molecular method to detect mutations in the MTB embB306 region.
Isola
Daniela
D
Dipartimento di Chirurgia e Scienze Odontostomatologiche, Università di Cagliari, Cagliari, Italy.
Pardini
Manuela
M
Varaine
Francis
F
Niemann
Stefan
S
Rüsch-Gerdes
Sabine
S
Fattorini
Lanfranco
L
Orefici
Graziella
G
Meacci
Francesca
F
Trappetti
Claudia
C
Rinaldo Oggioni
Marco
M
Orrù
Germano
G
D000995:Antitubercular Agents / Q000494:pharmacology*; D003062:Codon; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D024901:Drug Resistance, Multiple, Bacterial / Q000235:genetics; D004977:Ethambutol / Q000494:pharmacology*; D006801:Humans; D008826:Microbial Sensitivity Tests; D009169:Mycobacterium tuberculosis / Q000187:drug effects* / Q000235:genetics*; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide; D017422:Sequence Analysis, DNA / Q000379:methods*; D014376:Tuberculosis / Q000382:microbiology
D000995:Antitubercular Agents; D003062:Codon; D004269:DNA, Bacterial; D004977:Ethambutol