Li Shijun, Liu Ying, Wang Yue, Chen Hong, Liu Chunting, Wang Yi
Laboratory of Bacterial Infectious Disease of Experimental Center, Guizhou Provincial Center for Disease Control and Prevention, Guiyang 550004, People's Republic of China.
Laboratory of Guiyang Center for Animal Disease Control and Prevention, Guiyang 550081, People's Republic of China.
Infect Drug Resist. 2019 Jul 30;12:2343-2353. doi: 10.2147/IDR.S211644. eCollection 2019.
species is responsible for brucellosis in human and animals, which is still of public health, veterinarian, and economic concern in many regions of the world. Here, a novel molecular diagnosis assay, termed loop-mediated isothermal amplification coupled with nanoparticles-based lateral flow biosensor (LAMP-LFB), was developed and validated for simply, rapidly, and reliably detecting all spp. strains. A set of six primers was designed based on the -specific gene . The -LAMP results were visually reported by biosensor within 2 mins. A variety of bacterial strains representing several species, as well as several Gram-negative and Gram-positive bacterial species were used to determine the analytical sensitivity and specificity of the assay. Optimal LAMP conditions were 63°C for 40 mins, and the assay's sensitivity was found to be 100 fg of genomic DNA in the pure cultures. No cross-reactions to non- strains were obtained; thus, analytical specificity of LAMP-LFB assay is of 100%. Using the protocol, 20 mins for rapid DNA preparation followed by isothermal amplification (40 mins) combined with biosensor detection (2 mins) resulted in a total assay time of approximately 65 mins. In the case of 117 whole blood samples, 13 (11.11%) samples were -positive by LAMP-LFB, and the diagnostic accuracy was 100% when compared to the culture-biotechnical method. In conclusion, -LAMP-LFB technique developed in this study is a sensitive and specific method to rapidly identify all spp. strains, and can be applied as a potential diagnostic tool for brucellosis in basic, clinical, and field laboratories.
该物种可导致人类和动物感染布鲁氏菌病，在世界许多地区，这仍然是一个涉及公共卫生、兽医和经济的问题。在此，我们开发并验证了一种新型分子诊断方法，称为环介导等温扩增结合基于纳米颗粒的侧向流动生物传感器（LAMP-LFB），用于简单、快速且可靠地检测所有该物种的菌株。基于该物种特异性基因设计了一组六条引物。LAMP结果可在2分钟内通过生物传感器直观报告。使用了代表几种该物种的多种细菌菌株，以及几种革兰氏阴性和革兰氏阳性细菌物种来确定该检测方法的分析灵敏度和特异性。最佳LAMP条件为63°C孵育40分钟，该检测方法在纯培养物中的灵敏度为100 fg基因组DNA。未观察到与非该物种菌株的交叉反应；因此，LAMP-LFB检测方法的分析特异性为100%。使用该方案，快速DNA制备20分钟，随后进行等温扩增（40分钟）并结合生物传感器检测（2分钟），总检测时间约为65分钟。在117份全血样本中，13份（11.11%）样本经LAMP-LFB检测为该物种阳性，与培养生物技术方法相比，诊断准确率为100%。总之，本研究中开发的该物种LAMP-LFB技术是一种敏感且特异的方法，可快速鉴定所有该物种的菌株，可作为基础、临床和现场实验室中布鲁氏菌病的潜在诊断工具。
