Sayed Rafik Hamed, Soliman Rafik Twfik, Elsaady Shaimaa Abdelall
CLEVB, Agriculture Research Center, Cairo, Egypt.
Department of Microbiology, Faculty of Veterinary Medicine, Cairo University, Cairo, Egypt.
J Adv Vet Anim Res. 2023 Jun 30;10(2):292-300. doi: 10.5455/javar.2023.j681. eCollection 2023 Jun.
This work was conducted for the development of a 5-combi lateral flow immunochromatographic kit (LFK) for rapid and simultaneous identification of the common bacterial causes of bovine mastitis. The following pathogens are the identification targets of this kit: , , , , and in milk samples from suspected bovine mastitis cases. The conventional microbiological identification of these agents is not only time-consuming and requires a fully equipped laboratory but also requires experienced personnel.
Rabbit polyclonal antibodies (PAbs) specific to the antigenic components of the selected pathogens were prepared, and the pathogen-specific IgG was separated, purified, and conjugated with nanogold that was laid on the conjugate pad. Guinea pig PAbs specific to the microbial antigens of the selected pathogens were prepared, and their IgG content was separated, purified, and used as a capture antibody in the test (T) line on the nitrocellulose (NC) strips. Goat anti-rabbit IgG antibodies were used to capture the rabbit antibodies in the control (C) line of NC strips. The kit was held in a device comprising five strip-holding channels for the above-mentioned five bacterial species antigens. The developed LFK was evaluated, and its sensitivity and specificity were determined.
The developed kits were applied for the examination of bovine milk samples from suspected mastitis cases, and the average sensitivity, specificity, and accuracy of 5-combi LFK for the detection of the five selected bacterial species compared to bacteriological examination (gold standard test) were 93.90%, 80.83%, and 90.53%, respectively. The minimal microbial count that gave positive results using the developed LFK was 10 colony forming unit/ml. Treatment of the milk samples with an application buffer and its pre-incubation in trypticase soy broth for 6 h at 37°C before testing significantly increased the sensitivity of the prepared LFK. The developed kit proved simple and convenient, and the results could be obtained in less than 10 min.
开展一项工作以开发一种五联侧向流动免疫层析试剂盒（LFK），用于快速同时鉴定引起牛乳腺炎的常见细菌病因。以下病原体是该试剂盒的鉴定目标：[此处原文中病原体名称缺失]、[此处原文中病原体名称缺失]、[此处原文中病原体名称缺失]、[此处原文中病原体名称缺失]和[此处原文中病原体名称缺失]，来自疑似牛乳腺炎病例的牛奶样本。对这些病原体进行传统的微生物学鉴定不仅耗时，需要设备齐全的实验室，还需要经验丰富的人员。
制备针对所选病原体抗原成分的兔多克隆抗体（PAbs），分离、纯化病原体特异性IgG，并与铺在结合垫上的纳米金偶联。制备针对所选病原体微生物抗原的豚鼠PAbs，分离、纯化其IgG含量，并用作硝酸纤维素（NC）条上检测（T）线的捕获抗体。山羊抗兔IgG抗体用于捕获NC条对照（C）线中的兔抗体。该试剂盒置于一个包含五个条带固定通道的装置中，用于上述五种细菌抗原。对开发的LFK进行评估，并确定其灵敏度和特异性。
将开发的试剂盒应用于检测疑似乳腺炎病例的牛乳样本，与细菌学检查（金标准试验）相比，五联LFK检测所选五种细菌的平均灵敏度、特异性和准确性分别为93.90%、80.83%和90.53%。使用开发的LFK得到阳性结果的最低微生物计数为10菌落形成单位/毫升。在测试前，用应用缓冲液处理牛奶样本并在胰蛋白酶大豆肉汤中于37°C预孵育6小时，可显著提高所制备LFK的灵敏度。开发的试剂盒证明简单方便，不到10分钟即可获得结果。
