Batinga Maria Cryskely A, Dos Santos Jaíne C, Lima Julia T R, Bigotto Maria Fernanda D, Muner Kerstin, Faita Thalita, Soares Rodrigo M, da Silva David A V, Oliveira Trícia M F S, Ferreira Helena L, Diniz Jaqueline A, Keid Lara B
Departamento de Medicina Veterinária Preventiva e Saúde Animal, Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo, Pirassununga, Brazil.
Departamento de Medicina Veterinária, Faculdade de Zootecnia e Engenharia de Alimentos, Universidade de São Paulo, Pirassununga, SP, Brazil.
J Microbiol Methods. 2017 Dec;143:26-31. doi: 10.1016/j.mimet.2017.08.019. Epub 2017 Aug 31.
Brucella canis, a gram-negative, facultative intracellular and zoonotic bacterium causes canine brucellosis. Direct methods are the most appropriate for the detection of canine brucellosis and bacterial isolation from blood samples has been employed as gold-standard method. However, due to the delay in obtaining results and the biological risk of the bacterial culturing, the polymerase chain reaction (PCR) has been successfully used as an alternative method for the diagnosis of the infection. Sample preparation is a key step for successful PCR and protocols that provide high DNA yield and purity are recommended to ensure high diagnostic sensitivity. The objective of this study was to evaluate the performance of PCR for the diagnosis of B. canis infection in 36 dogs by testing DNA of whole blood obtained through different extraction and purification protocols. Methods 1 and 2 were based on a commercial kit, using protocols recommended for DNA purification of whole blood and tissue samples, respectively. Method 3 was an in-house method based on enzymatic lysis and purification using organic solvents. The results of the PCR on samples obtained through three different DNA extraction protocols were compared to the blood culture. Of the 36 dogs, 13 (36.1%) were positive by blood culturing, while nine (25.0%), 14 (38.8%), and 15 (41.6%) were positive by PCR after DNA extraction using methods 1, 2 and 3, respectively. PCR performed on DNA purified by Method 2 was as efficient as blood culturing and PCR performed on DNA purified with in-house method, but had the advantage of being less laborious and, therefore, a suitable alternative for the direct B. canis detection in dogs.
犬布鲁氏菌是一种革兰氏阴性、兼性细胞内寄生的人畜共患病细菌，可引起犬布鲁氏菌病。直接检测方法最适合用于犬布鲁氏菌病的检测，从血液样本中分离细菌一直被用作金标准方法。然而，由于获得结果的延迟以及细菌培养的生物风险，聚合酶链反应（PCR）已成功用作诊断感染的替代方法。样本制备是PCR成功的关键步骤，建议采用能提供高DNA产量和纯度的方案，以确保高诊断灵敏度。本研究的目的是通过检测经不同提取和纯化方案获得的全血DNA，评估PCR诊断36只犬感染犬布鲁氏菌的性能。方法1和方法2基于商业试剂盒，分别使用推荐用于全血和组织样本DNA纯化的方案。方法3是一种基于酶解和有机溶剂纯化的内部方法。将通过三种不同DNA提取方案获得的样本的PCR结果与血培养结果进行比较。在36只犬中，13只（36.1%）血培养呈阳性，而分别使用方法1、方法2和方法3提取DNA后，PCR检测呈阳性的犬分别有9只（25.0%）、14只（38.8%）和15只（41.6%）。用方法2纯化的DNA进行的PCR与血培养以及用内部方法纯化的DNA进行的PCR效果一样好，但具有操作更简便的优点，因此是直接检测犬体内犬布鲁氏菌的合适替代方法。
