To diagnose brucellosis effectively, many genus- and species-specific detection methods
based on PCR have been developed. With conventional PCR assays, real-time PCR techniques
have been developed as rapid diagnostic tools. Among them, real-time PCR using
hybridization probe (hybprobe) has been recommended for bacteria with high DNA homology
among species, with which it is possible to make an accurate diagnosis by means of an
amplification curve and melting peak analysis. A hybprobe for B. abortus
was designed from a specific single-nucleotide polymorphism (SNP) on the
fbaA gene. This probe only showed specific amplification of B.
abortus from approximately the 14th cycle, given a melting peak at 69°C. The
sensitivity of real-time PCR was revealed to be 20 fg/µl by 10-fold DNA
dilution, and the detection limit was 4 CFU in clinical samples. This real-time PCR showed
greater sensitivity than that of conventional PCR and previous real-time PCR based on
Taqman probe. Therefore, this new real-time PCR assay could be helpful for differentiating
B. abortus infection with rapidity and accuracy.
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