Comparative diagnostic evaluation of OMP31 gene based TaqMan® real-time PCR assay with visual LAMP assay and indirect ELISA for caprine brucellosis

Abstract

Brucellosis is one of the leading causes of abortion in domestic animals that imposes costs on both economy and society. The disease is highly zoonotic and poses risk to animal handlers due to its zoonotic nature. It causes stillbirth, loss of kids and abortion in last term of pregnancy. Reproductive damage includes infertility in does and orchitis and epididymitis in breeding bucks, which result in high financial losses to farmers and the agriculture industry as a whole. It requires highly sensitive and specific assays to diagnose the disease at field level. In the current study, a visual loop-mediated isothermal amplification (LAMP) assay and the TaqMan® real-time PCR were developed with high sensitivity and specificity. For the TaqMan® probe, real-time PCR primers were developed using Omp31 gene as target and primers were designed using discontiguous conserved sequences of Omp31 gene. The Omp31 probes were designed by attaching 6-FAM reporter dye at the 5' end and BHQ-1 quencher at the 3' end. Published primers were used for visual LAMP assay targeting the Omp25 gene. Sensitivity of the standardized visual LAMP assay and TaqMan® real-time PCR assay was determined by serial dilution of positive Brucella melitensis DNA (10 to 10 ng) obtained from standard culture. The TaqMan® probe real-time assay can detect as low as 100 fg of B. melitensis DNA, whereas culture from vaginal swab washings has a limit of detection (LOD) of only 1 cfu/ml. Similarly, the visual LAMP assay can detect as low as 10 fg of B. melitensis DNA as compared to an LOD of 30 cfu/ml from culture of vaginal swab washings. Both assays were compared with serological tests (serum tube agglutination test (STAT) and indirect enzyme-linked immunosorbent assay (iELISA)) for diagnostic sensitivity and specificity. Diagnostic sensitivities and specificities for TaqMan® real-time PCR vs. LAMP assays were 98 and 100% vs. 100 and 97.8%, respectively. Results of visual LAMP assay indicated that LAMP is a fast, specific, sensitive, inexpensive and suitable method for diagnosis of B. melitensis infection under field conditions. On the other hand, Omp31 TaqMan® probe real-time assay can be used in conjunction with the other field-based diagnostic tests due to its high specificity.

Multiplex SYBR green real time PCR for the simultaneous detection and differentiation of four important reproductive infectious pathogens

“…The arrival of the real-time PCR has offered an opportunity to meet the requirements for rapid diagnosis (Saini et al, 2017) PCR offers a better method for swift and sensitive detection of microorganism. On the downside conventional PCR usually consists of 3 phases: DNA isolation, PCR amplification, and post-PCR analysis, which includes gel electrophoresis, hybridization and/or sequencing.…”

Molecular detection of important abortion‐causing microorganisms in preputial swab of breeding bucks using PCR‐based assays

“…For ruling out the presence of Brucella melitensis in preputial washings of breeding bucks, OMP31‐TaqMan ® probe PCR was used as per the reaction mix and thermal conditions described by Saini et al. (2017)…”

“…For ruling out the presence of Brucella melitensis in preputial washings of breeding bucks, OMP31-TaqMan ® probe PCR was used as per the reaction mix and thermal conditions described by Saini et al (2017) For Campylobacter spp., the oligonucleotides used are F: 5´-T GTCGTCAGCTCGTGTCGTG-3´ and R: 5´-TCTACGATTACTAGCGATT CCG-3´in a final 20 μl reaction mix to amplify a 276 bp PCR product (Beena et al, 2017). While, the PCR was performed for Coxiella burnetii using the 10 picomole each primers trans1: 5-′TATGTATCCACCGTAGCCAGTC-3′ and trans2: 5-′CCCAACAACACCTCCTTATTC-3′ to generate a 687bp PCR amplicon (Berri, Laroucau, & Rodolakis, 2000).…”

PCR Based Molecular Diagnostic Assays for Brucellosis: A Review

“…Apart from the above discussed novel real time PCR assays, a lot of work has been done so far on the validation aspect of these real time PCR assays in different countries (Al Dahouk et al, 2007;Queipo-Ortuno et al, 2008;Amoroso et al, 2011;Doosti and Dehkordi, 2011;Kumar et al, 2015;Mukherjee et al, 2015;Awwad et al, 2016;Kumar et al, 2017;Saini et al, 2017 andPatel et al, 2018). All these studies have found real time PCR as fast, sensitive and reliable tool for early detection of causative organism in the biological samples so that control and eradication programmes can be adopted as early as possible to minimise the losses to animal husbandry sector.…”