Computational protocol: Differential diagnosis of Brucella abortus by real-time PCRbased on a single-nucleotide polymorphisms

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[…] Bacterial strains and DNA samples: A total of 296 Brucella strains were included: 22 Brucella reference strains; 110 Mongolian isolates (16 B. abortus and 94 B. melitensis); 156 Korean isolates (84 B. abortus and 72 B. canis); and 8 non-Brucella strains reported to be serologically cross-reactive or phylogenetically similar bacteria (). B. abortus and B. melitensis from Mongolia were provided by Institute of Veterinary Medicine (IVM), through a collaborative project conducted from 2012 to 2014. The isolates from Mongolia were B. abortus biovar (bv.) 3 (9 strains) and untype (7) and B. melitensis bv. 1 (67), bv. 3 (10) and Rev. 1 (17). In addition, all of the Korean B. abortus bv. 1 was obtained from slaughtered cattle with brucellosis beginning in 2008, and the B. canis was from dog-breeding farms during 2002–2011. All of the Brucella isolates were identified by the classical biotyping assay including colony morphology, lysis by phages, oxidase, urease activity, growth on dyes and agglutination with monospecific sera (anti-A, -M and -R) [] and also confirmed specific bands for Brucella species by the differential multiplex PCR []. Genomic DNA for real-time PCR was extracted using a Blood & Tissue kit (Qiagen Ltd., Seoul, South Korea) per the manufacturer’s instructions.Hybprobe design and real-time PCR conditions: To develop B. abortus-specific real-time PCR assays, comparative sequence analysis, using fbaA gene region in whole genome sequences and/or partial sequences of 22 Brucella reference strains, was performed with the CLC Main Workbench software program version 6.0 (Insillicogen Inc., Aarhus N, Denmark). Based on the new B. abortus-specific SNP sites, the primer and probe sets were designed and developed using BEACON designer (Sigma-Aldrich, St. Louis, MO, U.S.A.).Real-time PCR with hybprobe was performed using 4.0 µl of 5 × genotyping master mix, 0.5 µl of each primer, 0.3 µl of each hybprobe, 13.4 µl of D.W. and 1.0 µl of DNA in a 20 µl total volume. After centrifugation for the removal of bubbles from the PCR plate, amplification and melting curve analysis were conducted using a LightCycler® 480II (Roche Diagnostic, Mannheim, Germany). The real-time PCR amplification was performed with an initial denaturation step of 95°C for 5 min, followed by 45 cycles of 95°C for 10 sec, 65°C for 15 sec and 68°C for 15 sec. After amplification, melting analysis was performed at between 40°C and 80°C at a rate of increase of 0.1°C.Specificity and sensitivity of real-time PCR assay: The specificity of real-time PCR assay using 22 Brucella reference strains, Brucella isolates and non-Brucella bacteria was assessed (). Its sensitivity was determined from a DNA concentration of 1 ng/µl to 1 fg/µl by serial 10-fold dilution of the B. abortus 544 reference strain. DNA concentration was measured using a Nanodrop ND-1000 UV/UVS spectrophotometer (Nanodrop Tech., Wilmington, DE, U.S.A.). These results were compared with those of a 16S rRNA [] and BaSS-PCR assays [], which were used to identify Brucella species and B. abortus biovars 1, 2 and 4 conventionally.Detection limits of real-time PCR assay: To compare the analytical sensitivity of real-time PCR assay in the clinical specimens, artificial inoculation using a B. abortus strain in the clinical samples was conducted. Briefly, ten-fold serial dilutions of the B. abortus strain with 0.85% saline were processed into the macerated lymphoid tissue, and then, each spiked sample was cultivated on three tryptic soy agars and was calculated in colony-forming units (CFU). The DNA of the spiked samples was extracted using a commercial blood and tissue kit (Qiagen Ltd.) according to the manufacturer’s protocols.Evaluation of real-time PCR assay: To apply real-time PCR assay to the clinical specimens, twelve samples (supramammary, submandibular, inguinal and parotid lymph nodes, testicle and buffy coat) were acquired from seropositive Korean native cattle on a breeding farm (). These specimens were ground in 1 ml of PBS buffer and spread onto tryptic soy agar supplemented with 5% bovine serum (GIBCO, Grand Island, NY, U.S.A.) and 5% sheep blood agar for 3–4 days at 37°C and 5% CO2. Genomic DNA from 200 µl of ground sample was extracted using a Blood & Tissue kit (Qiagen Ltd.) per the manufacturer’s instructions and was submitted to real-time PCR assay. […]

Pipeline specifications

Software tools CLC Main Workbench, Beacon Designer
Applications WGS analysis, qPCR
Organisms Brucella abortus
Diseases Brucellosis, Infection