基于布鲁氏菌omp25基因的环介导等温技术建立及初步应用

Establishment and Application of Loop-Mediated Isothermal Amplification for Rapid Detection of Brucella

为了建立布鲁氏菌环介导等温技术(LAMP),并应用于基层布病诊断,本实验针对布鲁氏菌外膜蛋白基因Omp25区域设计6条引物,用SYBR Green I染料做指示剂,65℃反应60 min,85℃5 min终止,根据SYBR Green I颜色变化判定结果,用普通PCR验证LAMP检测技术的灵敏度和准确性,并对临床疑似布病病料进行检测验证其临床检测意义。结果显示:LAMP检测的灵敏度为4.2 fg/μL布鲁氏菌基因组DNA,比普通PCR高100倍左右;其特异性较好,能够准确检测布鲁氏菌,琼脂糖凝胶电泳显示阳性结果均为梯形条带,而大肠杆菌、卡介苗、金黄色葡萄球菌、李氏杆菌等均为阴性;对新疆4个地区25份绵羊流产胎儿病料进行LAMP和普通PCR方法平行检测,其符合率为100%。结论:LAMP检测技术用于布病临床诊断具有快速、高效、便捷等特点,为基层布病的病原学诊断具有潜在的实用价值。

The aim of this research is to set up a protocol of loop-mediated isothermal amplification(LAMP) and apply it to the grass-roots level detection of Brucella.Six pairs of primers specific to the region of Omp25 gene were designed.The experiment reaction was performed in a single tube at 65 ℃ for 60 min with the addition of SYBR Green I after the reaction,and the result was determined by the color change of SYBR Green I.Additionally,the sensitivity and accuracy of this method were evaluated with ordinary PCR.The clinical specimens were determined by LAMP assay.The results showed that the sensitivity of this assay was about 4.2 fg/μL genomic DNA,which was about 100 times higher than ordinary PCR method.Meanwhile,the accuracy of LAMP assay showed that this method was specific for detecting brucella spp.,and no reactivity were found with Escherichia coli,Bacillus Calmette Guerin,Staphylococcus aureus and Listeria spp.The LAMP assay was evaluated with 25 aborted sheep fetuses from four brucellosis epidemic areas in Xinjiang.The results showed that the coincident rate was 100% between LAMP assay and ordinary PCR,which suggesting that the LAMP assay is a useful tool due to its more sensitivity and accuracy,and has potential practical value in the diagnosis of brucellosis.