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 singlenucleotide 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.
Highlights
Brucellosis is known as a major zoonotic disease that can cause reproductive problems, such as abortion, stillbirth or infertility in livestock and wild animals [1,2]
All of the Korean B. abortus bv. 1 was obtained from slaughtered cattle with brucellosis beginning in 2008, and the B. canis were from dog-breeding farms during 2002-2011
B. abortus-specific singlenucleotide polymorphism (SNP) were detected at the fbaA gene of B. abortus chromosome II (Genbank accession No AE 017224), with cytosine changed to thymine at 360432 on B. abortus chromosome II
Summary
Brucellosis is known as a major zoonotic disease that can cause reproductive problems, such as abortion, stillbirth or infertility in livestock and wild animals [1,2]. The genus Brucella consists of ten species; six species (Brucella abortus, B. melitensis, B. suis, B. canis, B. ovis and B. neotomae) considered classic members and four species (B. ceti, B. pinnipedialis, B. microti, and B. inopinata) considered atypical types of Brucella. Classification of Brucella species has been mainly based on host preferences and classical phenotypic biotyping [2,3]. In terms of the diagnosis of brucellosis, serological assays and bacterial cultivation have mainly been used. Serologic methods are very sensitive and rapid methods to perform, but sometimes falsepositive reactions occur with cross-reactive bacteria, such as Yersinia enterocolitica O:9, due to the similar structure of the O-chain in the smooth lipopolysaccharide portion [4,5]. Bacterial culture is considered a 'gold standard' with high specificity, but it is timeconsuming and requires a highly trained workforce and a wellequipped laboratory due to the biohazard risks with Brucella [6]
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