Evaluation of PCR methods for detection of Brucella strains from culture and tissues

Abstract

The genus Brucella causes significant economic losses due to infertility, abortion, stillbirth or weak calves, and neonatal mortality in livestock. Brucellosis is still a zoonosis of public health importance worldwide. The study was aimed to optimize and evaluate PCR assays used for the diagnosis of Brucella infections. For this aim, several primers and PCR protocols were performed and compared with Brucella cultures and biological material inoculated with Brucella. In PCR assays, genus- or species-specific oligonucleotide primers derived from 16S rRNA sequences (F4/R2, Ba148/928, IS711, BruP6-P7) and OMPs (JPF/JPR, 31ter/sd) of Brucella were used. All primers except for BruP6-P7 detected the DNA from reference Brucella strains and field isolates. In spiked blood, milk, and semen samples, F4-R2 primer-oriented PCR assays detected minimal numbers of Brucella. In spiked serum and fetal stomach content, Ba148/928 primer-oriented PCR assays detected minimal numbers of Brucella. Field samples collected from sheep and cattle were examined by bacteriological methods and optimized PCR assays. Overall, sensitivity of PCR assays was found superior to conventional bacteriological isolation. Brucella DNA was detected in 35.1, 1.1, 24.8, 5.0, and 8.0% of aborted fetus, blood, milk, semen, and serum samples by PCR assays, respectively. In conclusion, PCR assay in optimized conditions was found to be valuable in sensitive and specific detection of Brucella infections of animals.

Evaluation of designed IS711 primers and universal primers of B4 and B5 for detection of Brucella spp. in clinical samples

“…It is expected that IS711 target is much more sensitive, since there are several copies of IS711 but a single copy of BCSP31 gene in the Brucella genome. The designed IS711 specific primer could detect at least 1×10 1 cfu/ ml bacteria in the samples, approximately 5×10 1 times more sensitive than the other reported IS711 primers that were used in the detection of this gene by Ciftciin, which could detect 5×10 2 cfu/ml bacteria [26]. These achievements confirm the PCR results that were reported by Khosravi [39] and Elfaki [40].…”

“…The optimization has direct effects on the diagnostic outcomes [25,26]. Furthermore, according to the previous reports molecular techniques such as PCR are known as a helpful option for detection of fastidious bacteria like Brucella [18].…”

“…Generally, the Brucella culture remains the most specific diagnostic method for brucellosis, which is the most sensitive method in the acute and sub-acute phases of the diseases [25,26]. The serological techniques lack sensitivity and specificity due to antigenic cross-reaction and may lead to erroneous diagnosis, especially when antibody titers are lower than 160.…”

Evaluation of designed IS711 primers and universal primers of B4 and B5 for detection of Brucella spp. in clinical samples

“…Generally, the conventional classic diagnostic methods for detection of brucellosis have low sensitivity and specificity, with a high risk of incorrect diagnosis which may lead to mistreatment [25,26]. In contrast, the advanced molecular diagnostic tools are a reliable methodology to gain accurate and sharp results within a short time.…”

Detection of Brucella abortus by a platform functionalized with protein A and specific antibodies IgG

“…Bacterial isolation from several tissues or body fluids may be influenced by culture methods, low number of bacteria, and previous use of antibiotics, time of sample collection, and stage of the disease (Espinosa et al, ). Instead, serologic and molecular techniques are used as supporting methods of diagnosis (Araj, ; Çiftci et al, ; Geresu & Kassa, ; Gómez et al, ). Most of the immunologic diagnostic methods have poor specificity since many antigenic structures of Brucella spp.…”