Departamento de Medicina Veterinária Preventiva e Saúde Animal da Faculdade de Medicina Veterinária e Zootecnia da Universidade de São Paulo, São Paulo, SP, Brazil.
Departamento de Medicina Veterinária da Faculdade de Zootecnia e Engenharia de Alimentos da Universidade de São Paulo, São Paulo, SP, Brazil.
Mol Cell Probes. 2018 Jun;39:1-6. doi: 10.1016/j.mcp.2018.02.003. Epub 2018 Mar 7.
Canine brucellosis is caused by Brucella canis, a gram negative and facultative intracellular bacterium that is commonly associated with reproductive failures in dogs. The accurate diagnosis of the infection relies on the use of serological tests associated with blood culturing to guarantee sensitivity. The polymerase chain reaction (PCR) can replace the culturing procedure for the direct diagnosis of the infection because of its speed, high specificity and sensitivity values; however, it depends on some laboratory infrastructure to be conducted. The loop-mediated isothermal amplification (LAMP) may be an alternative method for DNA amplification in a shorter period, using simpler equipment, and with a lower cost. This study evaluated the potential of molecular tools based on PCR and LAMP using primers targeting the insertion sequence IS711 for Brucella detection in three groups of dogs (infected, non-infected and suspected of brucellosis), which were determined according to the results of blood culturing and clinical examination. The performance of the three diagnostic tests was also determined using McNemar test and Kappa coefficient. The proportion of positive samples detected by blood culturing, PCR and LAMP was respectively 31.57% (18/57), 33.34% (19/57), and 14.03% (8/57). The agreement between blood culturing and PCR was almost perfect, while the agreement of PCR and blood culturing compared to LAMP was fair. The diagnostic sensitivity of PCR and LAMP was respectively 100% (18/18) and 44.44% (8/18), while the diagnostic specificity of both tests was 100% (21/21). LAMP performance was not satisfactory for canine brucellosis diagnosis because of the low diagnostic sensitivity of the test. The IS711 based PCR, otherwise, showed high values of sensitivity and specificity, which makes it a good alternative for use for the rapid diagnosis of canine brucellosis.
犬布鲁氏菌病是由布鲁氏菌属犬种引起的，这种革兰氏阴性兼性细胞内细菌通常与犬只生殖失败有关。感染的准确诊断依赖于血清学检测与血液培养相结合的方法，以保证其敏感性。聚合酶链反应（PCR）可以替代培养程序，直接诊断感染，因为它速度快、特异性和敏感性高；然而，它需要一些实验室基础设施才能进行。环介导等温扩增（LAMP）可以在更短的时间内使用更简单的设备和更低的成本替代 DNA 扩增的方法。本研究评估了基于 PCR 和 LAMP 的分子工具的潜力，使用针对插入序列 IS711 的引物检测三组犬（感染、未感染和疑似布鲁氏菌病）中的布鲁氏菌，这三组犬是根据血液培养和临床检查结果确定的。还使用 McNemar 检验和 Kappa 系数确定了三种诊断试验的性能。血液培养、PCR 和 LAMP 检测的阳性样本比例分别为 31.57%（18/57）、33.34%（19/57）和 14.03%（8/57）。血液培养和 PCR 之间的一致性几乎是完美的，而 PCR 与血液培养之间的一致性与 LAMP 相比是公平的。PCR 和 LAMP 的诊断敏感性分别为 100%（18/18）和 44.44%（8/18），而两种检测方法的诊断特异性均为 100%（21/21）。LAMP 对犬布鲁氏菌病的诊断性能并不令人满意，因为该试验的诊断敏感性较低。基于 IS711 的 PCR 则具有较高的敏感性和特异性，是犬布鲁氏菌病快速诊断的良好替代方法。
