Li Chao, Shi Yaoqiang, Yang Guangying, Xia Xue-Shan, Mao Xiaoqin, Fang Yue, Zhang A-Mei, Song Yuzhu
Molecular Diagnosis Laboratory, Faculty of Life Science and Technology, Kunming University of Science and Technology, Kunming, Yunnan 650500, P.R. China.
Molecular Medicine Center of Yunnan Province, Kunming, Yunnan 650500, P.R. China.
Exp Ther Med. 2019 Jan;17(1):131-136. doi: 10.3892/etm.2018.6910. Epub 2018 Oct 31.
is one of the three most pathogenic bacteria that frequently cause life-threatening opportunistic human infections, pneumonia, and lower respiratory tract infections in immunocompromised hosts, particularly in the burns ward. The present study aimed to establish a loop-mediated isothermal amplification (LAMP) method for the rapid and sensitive detection of -specific gene (GenBank ID: 882161). The gene was obtained through local and online BLAST, and specific primers were designed for this gene. Reaction conditions were optimized at 65°C for 30 min and 80°C for 2 min, whereas the reaction system contained 5.2 mM Mg, 8 U Bst 2.0 DNA polymerase, 1.4 mM deoxyribonucleotide and 0.2 and 1.6 µM of the outer and inner primers, respectively. The LAMP method was evaluated using 150 and 170 non- strains. Positive reactions were observed on 150 strains, whereas all non- strains exhibited negative results. Plasmids with the specific gene and mouse blood with were used for sensitivity assay. The detection limit of LAMP was 1 bacterium/reaction. Results indicated that the LAMP method targeted to is a fast, specific, sensitive, inexpensive and suitable method for detection of .
是三种最具致病性的细菌之一,经常在免疫功能低下的宿主中引起危及生命的机会性人类感染、肺炎和下呼吸道感染,尤其是在烧伤病房。本研究旨在建立一种环介导等温扩增(LAMP)方法,用于快速、灵敏地检测 -特异性基因(GenBank ID:882161)。通过本地和在线BLAST获得该基因,并针对该基因设计特异性引物。反应条件在65°C优化30分钟,80°C优化2分钟,而反应体系分别包含5.2 mM Mg、8 U Bst 2.0 DNA聚合酶、1.4 mM脱氧核苷酸以及0.2和1.6 µM的外引物和内引物。使用150株 和170株非 菌株对LAMP方法进行评估。在150株 菌株上观察到阳性反应,而所有非 菌株均呈现阴性结果。使用含有特异性基因的质粒和含有 的小鼠血液进行灵敏度测定。LAMP的检测限为1个细菌/反应。结果表明,针对 的LAMP方法是一种快速、特异、灵敏、廉价且适合检测 的方法。