Differential diagnosis of Brucella abortus by real-time PCR based on a single-nucleotide polymorphisms
Ji-Yeon KIM, Sung-Il KANG, Jin Ju LEE, Kichan LEE, So-Ra SUNG, Janchivdorj ERDENEBAATAAR,
Batbaatar VANAABAATAR, Suk Chan JUNG, Yong Ho PARK, Han-Sang YOO and Moon HER
ABSTRACT. 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.
KEY WORDS: Brucella abortus, fbaA gene, hybprobe, real-time PCR, SNP
doi: 10.1292/jvms.15-0541; J. Vet. Med. Sci. 78(4): 557–562, 2016