Division of Animal Health, ICAR-Central Sheep and Wool Research Institute, Avikanagar, India.
Division of Biological Products, ICAR-Indian Veterinary Research Institute, Izatnagar, India.
J Microbiol Methods. 2021 May;184:106185. doi: 10.1016/j.mimet.2021.106185. Epub 2021 Mar 5.
The widely used serodiagnostic test (RBPT, CFT, I-ELISA and FPA) for diagnosis of brucellosis cannot detect vertically infected or carrier animals that are seronegative, a persistent source of infection to other susceptible animals in the herd. For reducing transmission of disease within the herd, these animals must be detected using a rapid, sensitive, user friendly penside diagnostic test. In the present study, Lateral Flow immunoassay (LFA) strip test was developed for detection of Brucellaspp. from clinical samples (bovine aborted foetal stomach contents). The LFA strip was fabricated by printing anti-Brucella polyclonal antibodies (PAb) and anti-bovine antibodies IgG on test and control line, respectively. For conjugation, colloidal gold nanoparticles (30 nm GNP, Sigma, USA) were conjugated with anti-brucella PAb. The LFA strip test was able to detect 10 cfu/ml B.abortus S99 inactivated organism in PBS and it did not exhibit any cross reactivity with selected non Brucella pathogens. To further validate, 115 clinical specimens were tested using LFA strip test. The relative sensitivity (DSn) and relative specificity (DSp) of LFA strip test was determined by ROC curve analysis using PCR and culture method as reference standard. DSn and DSp of LFA strip test was observed as 78.57% (95%CI: 49.2-95.3); 93.07% (95%CI: 86.2-97.2) and 80.0% (95%CI:51.9-95.7); 94.0% (95%CI:0.795-0.925) using culture and PCR as reference diagnostic tests, respectively. It may be concluded that, the LFA strip test can be used as a rapid penside diagnostic test for screening of brucellosis. To the best of our knowledge, this is the first report on development of GNP based LFA strip test for detection of Brucella spp. from bovine aborted fetal content samples.
用于诊断布鲁氏菌病的广泛使用的血清学诊断检测（RBPT、CFT、I-ELISA 和 FPA）无法检测到垂直感染或呈血清阴性的携带者动物，这些动物是畜群中其他易感动物持续感染的来源。为了降低畜群内疾病的传播，必须使用快速、敏感、用户友好的笔式诊断检测来检测这些动物。在本研究中，开发了用于从临床样本（牛流产胎儿胃内容物）中检测布鲁氏菌属 spp.的侧向流动免疫测定（LFA）条检测法。LFA 条通过分别在测试线和控制线打印抗布鲁氏菌多克隆抗体（PAb）和抗牛 IgG 抗体来制备。对于缀合，胶体金纳米颗粒（30nm GNP，Sigma，USA）与抗布鲁氏菌 PAb 缀合。LFA 条检测法能够检测到 PBS 中 10cfu/ml 的 B.abortus S99 灭活菌，并且与选定的非布鲁氏菌病原体没有任何交叉反应。为了进一步验证，使用 LFA 条检测法检测了 115 份临床标本。通过使用 PCR 和培养方法作为参考标准的 ROC 曲线分析，确定了 LFA 条检测法的相对灵敏度（DSn）和相对特异性（DSp）。使用培养和 PCR 作为参考诊断检测，LFA 条检测法的 DSn 和 DSp 分别为 78.57%（95%CI：49.2-95.3）；93.07%（95%CI：86.2-97.2）和 80.0%（95%CI：51.9-95.7）；94.0%（95%CI：0.795-0.925）。可以得出结论，LFA 条检测法可用于布鲁氏菌病的快速笔式诊断检测。据我们所知，这是首次报道基于 GNP 的 LFA 条检测法用于从牛流产胎儿内容物样本中检测布鲁氏菌属 spp.。
