Open AccessDevelopment and Evaluation of a Novel Single-Nucleotide-Polymorphism Real-Time PCR Assay for Rapid Detection of Fluoroquinolone-Resistant Mycoplasma bovis
Author(s) -
M. Ben Shabat,
Inna Mikula,
I. Gerchman,
Inna Lysnyansky
Publication year - 2010
Publication title -
journal of clinical microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.349
H-Index - 255
eISSN - 1070-633X
pISSN - 0095-1137
DOI - 10.1128/jcm.00699-10
Subject(s) - taqman , biology , mycobacterium bovis , real time polymerase chain reaction , mycoplasma , single nucleotide polymorphism , microbiology and biotechnology , virology , genotype , polymerase chain reaction , mycobacterium tuberculosis , genetics , tuberculosis , medicine , gene , pathology
Monitoring of the susceptibility ofMycoplasma bovis field isolates to antibiotics is important for the appropriate choice of treatment. However,in vitro susceptibility testing of mycoplasmas is technically demanding and time-consuming, especially for clinical isolates, and is rarely performed in mycoplasma diagnostic laboratories. Thus, the development of methods allowing rapid real-time detection of resistant strains ofM. bovis in clinical samples is a high priority for successful treatment. In this study, a novel TaqMan single-nucleotide-polymorphism (SNP) real-time PCR assay, which enables the rapid identification ofM. bovis strains with different susceptibilities to fluoroquinolones, was developed and evaluated. The TaqMan SNP real-time PCR assay is based on the amplification of a 97-bp fragment of theparC quinolone resistance-determining region (QRDR) and allows the specific detection of four possible genotypes: GAC or GAT (susceptible to fluoroquinolones) and AAC or AAT (resistant to fluoroquinolones). Four TaqMan minor groove binder (MGB) probes identifying 1-base mismatches were designed and applied in a dual-probe assay with two reaction tubes. The TaqMan SNP real-time PCRs developed are highly specific forM. bovis , with a detection limit of 5 fg/μl (about 5M. bovis genomes). In addition, all four SNP real-time PCR tests have almost the same efficiency (97.7% [GAC], 94% [AAC], 99.99% [GAT], and 98% [AAT]). Taken together, the data suggest that this SNP real-time PCR assay has potential as a routine diagnostic test for the detection of decreased susceptibility ofM. bovis to fluoroquinolones.