Yang Xinggui, Wang Yue, Liu Ying, Huang Junfei, Tan Qinqin, Ying Xia, Hu Yong, Li Shijun
Guizhou Provincial Center for Disease Control and Prevention, Guiyang, China.
School of Public Health, the Key Laboratory of Environmental Pollution Monitoring and Disease Control, Ministry of Education, Guizhou Medical University, Guiyang, China.
Front Bioeng Biotechnol. 2021 Nov 18;9:758564. doi: 10.3389/fbioe.2021.758564. eCollection 2021.
(), an important zoonotic pathogen in spp., is the major causative agent of abortion in cattle (namely, bovine brucellosis). Currently, although the isolation and identification of the were commonly accepted as the gold standard method, it cannot meet the requirements for early diagnostic strategies. Conventional PCR techniques and immunological tests can realize rapid detection of , but the demands for PCR thermal cyclers and/or specific antibodies hinder their application in basic laboratories. Thus, rapid, sensitive, and specific diagnostic strategies are essential to prevent and control the spread of the bovine brucellosis. In this work, a novel detection method for the rapid identification of , which uses loop-mediated isothermal amplification (LAMP) combined with a label-based polymer nanoparticles lateral flow immunoassay biosensor (LFIA), was established. One set of specific -LAMP primers targeting the gene was designed by the online LAMP primer design tool. The -LAMP-LFIA assay was optimized and evaluated using various pathogens and whole blood samples. The optimal amplification temperature and time for -LAMP-LFIA were determined to be 65°C and 50 min, respectively. The -LAMP-LFIA method limit of detection (LoD) was 100 fg per reaction for pure genomic DNA of . Meanwhile, the detection specificity was 100%, and there was no cross-reactivity for other members and non- strains. Furthermore, the entire procedure, including the DNA preparation for whole blood samples (30 min), isothermal incubation (50 min), and LFIA detection (2-5 min), can be completed in approximately 85 min. Thus, the -LAMP-LFIA assay developed was a simple, rapid, sensitive, and reliable detection technique, which can be used as a screening and/or diagnostic tool for in the field and basic laboratories.
布鲁氏菌属中的一种重要人畜共患病原体，是牛流产的主要致病因子（即牛布鲁氏菌病）。目前，尽管布鲁氏菌的分离鉴定被普遍认为是金标准方法，但它无法满足早期诊断策略的要求。传统的聚合酶链反应（PCR）技术和免疫检测可以实现布鲁氏菌的快速检测，但对PCR热循环仪和/或特异性抗体的需求阻碍了它们在基层实验室的应用。因此，快速、灵敏且特异的诊断策略对于预防和控制牛布鲁氏菌病的传播至关重要。在这项工作中，建立了一种用于快速鉴定布鲁氏菌的新型检测方法，该方法采用环介导等温扩增（LAMP）结合基于标记的聚合物纳米颗粒侧向流动免疫分析生物传感器（LFIA）。通过在线LAMP引物设计工具设计了一组针对布鲁氏菌基因的特异性布鲁氏菌-LAMP引物。使用各种病原体和全血样本对布鲁氏菌-LAMP-LFIA检测进行了优化和评估。确定布鲁氏菌-LAMP-LFIA的最佳扩增温度和时间分别为65°C和50分钟。布鲁氏菌-LAMP-LFIA方法对布鲁氏菌纯基因组DNA的检测限为每个反应100 fg。同时，检测特异性为100%，对其他布鲁氏菌属成员和非布鲁氏菌菌株无交叉反应。此外，整个过程，包括全血样本的DNA制备（30分钟）、等温孵育（50分钟）和LFIA检测（2 - 5分钟），大约85分钟即可完成。因此，所开发的布鲁氏菌-LAMP-LFIA检测是一种简单、快速、灵敏且可靠的检测技术，可作为现场和基层实验室布鲁氏菌的筛查和/或诊断工具。
