Samples

One isolate of B. abortus (field strain MKD-1027), derived from culture collection of the Faculty of Veterinary Medicine-Skopje, and organs from 11 slaughtered seropositive ruminants (Table 3) were used for extraction of genomic DNA. Organs from one animal (spleen, supramammary, iliac and inguinal lymph nodes) were grinded and macerated together in a form of suspension suitable for cultivation, inoculated on selective (Farrell’s) media and archived at -80°C as “tissue sample”. Eleven tissue samples used in the study were randomly selected from the FVMS’s collection of tissue samples kept at -80°C.

Table 1. Cycling conditions for amplification of VNTR loci for Brucella genotyping

Table 2. Tenfold dilutions of B. abortus (MKD-1027) DNA with appropriate amounts of DNA in reaction and obtained Cq values (tested in duplicate)

Table 3. Clinical samples in which Brucella DNA was detected by IS711 rt-PCR and MLVA-16 profiles were determined using the described protocols

DNA extraction

Pure bacterial DNA used for implementation and optimization of the test protocols for detection and molecular typing was obtained from the MKD-1027 isolate using PureLink Genomic DNA kit (Life Technologies, USA), according to the manufacturer’s instructions for Gram negative bacteria. Concentration and purity of extracted bacterial DNA was determined spectrophotometrically (A₂₆₀ and A₂₈₀) using NanoDrop 2000c (Thermo Scientific, USA). Total genomic DNA was extracted from tissue samples using the same commercial kit, according to the manufacturer’s instructions for tissues. The genomic DNA extracted from tissue samples was used to evaluate the performance of implemented testing scheme.

Real time PCR

Genomic DNA from field strain MKD-1027 was used for optimization and performance evaluation of implemented real time PCR for Brucella spp. detection. Primers and probe used in the test are targeting IS711 and are described elsewhere (11). Eight tenfold dilutions corresponding to a DNA concentration range between 1.7 ng/µL and 0.17 fg/µL were prepared from the purified DNA sample and tested in duplicate. Real time PCR reaction was prepared in a final volume of 25 µL using 12.5 µL 2x RT PCR reaction mix for probes (Bio-Rad, USA), 8 µL nuclease-free water, 2 µL primer-probe mix and 2.5 µL of DNA sample. Final concentrations of primers and probe were 0.5 µM and 0.2 µM, respectively. Amplification was performed in IQ5 Thermal Cycler (Bio-Rad, USA) under the following conditions: initial denaturation at 95°C for 5 min and 42 cycles of denaturation at 95°C for 15 s, and combined annealing-extension at 60°C for 30 s. Efficacy of the reaction (E), coefficient of determination (R2) and slope of the standard curve were calculated using IQ5 software. One of the DNA dilutions with high quantification cycle (Cq) value has been defined as a future “positive control sample” and was independently tested ten times. Obtained Cq values were used for calculation of coefficient of variation (CV) standing for inter-assay reproducibility and upper and lower limits of the positive control (95% and 99% confidence levels).

MLVA-16 genotyping

For achieving the best analytical sensitivity and repeatability of the capillary electrophoresis system used, all loci from the MLVA-16 panel (22, 24) were amplified in singleplex PCR reactions using 5’ - fluorescently (6-FAM) labeled forward primers and Taq PCR Master mix kit(Qiagen, USA). Depending on the amount of input DNA, three different protocols for PCR reaction set-up and cycling were used (Table 1).

PCR reactions were set in a total volume of 15 µL (PCR-1 and PCR-2) and 25 µL (PCR-3), containing 1 x Qiagen master mix and 0.5 µM of each primer. All PCR amplifications were performed in a TC412 thermal cycler (Techne, UK).

The size of the PCR products was determined using capillary electrophoresis on ABI 310 system (Applied Biosystems, USA). Briefly, undiluted (PCR-3) or diluted (PCR-1, PCR-2) products have been prepared for electro kinetic injection by adding deionized formamide (Applied Biosystems, USA) and GS LIZ500 or GS LIZ1200 (Applied Biosystems, USA) size standards. Amplified loci from referent B. abortus S19 and B. melitensis Rev-1 strains were used as a control strains. Fragment sizing was performed by GeneMapper software ver. 4.0, according to a generated standard curve. Obtained raw data for the size of each locus was recorded in an excel sheet. Measured fragment size was corrected for the previously determined measuring aberration of the instrument. Corrected values were converted in tandem repeat units using Table for allele assignment freely available at MLVA Net for Brucella website (http://mlva.u-psud.fr/brucella/spip.php?article93). Aberration in size measuring was determined for each locus by sequencing. Complete sequences of all 16 loci for referent B. abortus S19 and B. melitensis Rev-1 strains were obtained using ABI Prism Big Dye Terminator (v3.1) cycle sequencing ready reaction kit (Applied Biosystems, USA).

Real time PCR – culture dilutions

Bacterial DNA was detected in seven out of eight tenfold DNA dilutions when tested with the real time PCR assay for detection of Brucella spp., corresponding to analytical sensitivity of 4.2 fg bacterial DNA (Fig. 1, Table 2). The implemented test protocol amplified broad range of DNA concentrations (4.2 ng to 4.2 fg) with excellent linearity (R2= 0.997) and 85.5% reaction efficacy (Fig. 2).

Figure 1 Amplification curves of B. abortus (MKD-1027) DNA (log view). Tenfold dilutions (from 4.2 ng to 0.42 fg) were tested with the implemented IS711 real time PCR. Fluorescence data is baseline subtracted

Figure 2 Standard curve obtained after testing tenfold dilutions of B. abortus (MKD-1027) DNA with the implemented IS711 real time PCR

Analysis of Cq values obtained in ten independent tests of 10-6 B. abortus (MKD-1027) DNA dilution, indicated very good reproducibility and inter-assay CV of 1.8%. This DNA dilution has been defined as a positive control for all future runs, with upper and lower Cq limits in the following range: 33 – 34.2 (95% confidence level) and 32.5 – 34.7 (99% confidence level) (data not shown).

MLVA-16 genotyping

The modified PCR protocol (PCR-3) used for amplification, combined with fluorescence based capillary electrophoresis system for detection, enabled detection of all loci from the MLVA-16 scheme, even in the highest real time PCR positive dilution (10-7) of B. abortus DNA (Fig. 3). Next dilution (10-8) was negative, meaning that the lower limit of detection of the implemented MLVA-16 typing system was equal to the lower limit of detection observed in the implemented real time PCR for detection of Brucella spp. However, PCR-3 protocol should be used only for samples with minor DNA amounts (close to the lower limit of detection), which could not be genotyped using agar-gel electrophoresis. Otherwise, unspecific fragments are likely to appear (Fig. 4), especially if DNA is extracted from clinical samples. The appearance of unspecific fragments could compromise the interpretation of results in situations when their size is very close to the size of existing alleles.

Figure 3 Electropherograms of the products obtained after two different PCR amplifications of bruce30 locus in highest detectable dilution (10-7) of B. abortus DNA. Amplified locus was detected only with the modified (PCR-3) protocol (upper electropherogram)

Figure 4 Electropherograms of the products obtained after two different PCR amplifications of bruce30 locus in 10-6 dilution of B. abortus DNA. Amplified locus was detected with both PCR protocols, but unspecific fragments were present when modified (PCR-3) protocol was used (lower electropherogram)

Real time PCR and genotyping - clinical samples

Brucella spp. DNA was detected in the eleven samples of genomic DNA extracted from the tissues. Obtained Cq values varied, indicating different Brucella concentrations in the tested samples (Table 3). Brucella DNA was detected in seven tissues from which Brucella could not be cultivated, although (as expected) with higher Cq values. Finally, MLVA-16 profiles were successfully obtained for all samples. There were some reproducibility problems when sample with highest Cq value (1-AL) was tested – some of the loci required more than one attempt for obtaining visible signal. This could be explained with low amount of DNA in the 1-AL sample, which was very close to the lower limit of detection of the system.