Yan Muxia, Li Weidong, Zhou Zhenwen, Peng Hongxia, Luo Ziyan, Xu Ling
Department of Haematology, Guangzhou Women and Children's Medical Centre, Guangzhou Medical University, Guangzhou 510623, China.
PICU and Neonatology, Xiao Lan Hospital of Southern Medical University, Guangzhou 510180, China.
Microb Pathog. 2017 Jan;102:1-7. doi: 10.1016/j.micpath.2016.10.025. Epub 2016 Nov 9.
In this work, loop-mediated isothermal amplification based detection assay using bacterial culture and bacterial colony for various common pathogens direct detection had been established, evaluated and further applied. A total of five species of common pathogens and nine detection targets (tlh, tdh and trh for V. Parahaemolyticus, rfbE, stx1 and stx2 for E. coli, oprI for P. aeruginosa, invA for Salmonella and hylA for L. monocytogenes) were performed on bacterial culture and bacterial colony LAMP. To evaluate and optimize this assay, a total of 116 standard strains were included. Then, for each detected targets, 20 random selected strains were applied. Results were determined through both visual observation of the changed color by naked eye and electrophoresis, which increased the accuracy of survey. The minimum adding quantity of each primer had been confirmed, and the optimal amplification was obtained under 65 °C for 45 min with 25 μl reaction volume. The detection limit of bacterial culture LAMP and PCR assay were determined to be 10 and 10 or 10 CFU/reaction, respectively. No false positive amplification was observed when subjecting the bacterial -LAMP assay to 116 reference strains. This was the first report of colony-LAMP and culture-LAMP assay, which had been demonstrated to be a fast, reliable, cost-effective and simple method on detection of various common pathogens.
在本研究中，建立、评估并进一步应用了基于环介导等温扩增技术的检测方法，该方法利用细菌培养物和菌落直接检测多种常见病原体。对副溶血性弧菌的tlh、tdh和trh、大肠杆菌的rfbE、stx1和stx2、铜绿假单胞菌的oprI、沙门氏菌的invA以及单核细胞增生李斯特菌的hylA这5种常见病原体的9个检测靶点进行了细菌培养和菌落环介导等温扩增检测。为评估和优化该检测方法，共纳入了116株标准菌株。然后，针对每个检测靶点，随机选取20株菌株进行检测。通过肉眼观察颜色变化和电泳两种方式确定结果，提高了检测的准确性。确定了各引物的最低添加量，在65℃、25μl反应体积下扩增45分钟可获得最佳扩增效果。细菌培养环介导等温扩增检测和聚合酶链反应检测的检测限分别确定为10 CFU/反应和10或10 CFU/反应。对116株参考菌株进行细菌环介导等温扩增检测时未观察到假阳性扩增。这是关于菌落环介导等温扩增检测和培养环介导等温扩增检测的首次报道，已证明该方法是一种快速、可靠、经济高效且简便的多种常见病原体检测方法。
