Huang Simo, Yang Zhan, Zou Dayang, Dong Derong, Liu Anheng, Liu Wei, Huang Liuyu
Institute of Disease Control and Prevention, Academy of Military Medical Sciences, Beijing, P. R. China.
J Med Microbiol. 2016 Aug;65(8):760-769. doi: 10.1099/jmm.0.000300. Epub 2016 Jun 23.
Fusobacterium nucleatum is associated with various human diseases such as periodontal disease and colorectal cancer (CRC); thus, F. nucleatum detection might serve as a novel diagnostic tool. Here, we describe the development of a sensitive and rapid molecular method for detecting two F. nucleatum genes: the highly conserved nusG and fadA, which encode a critical host colonization factor. Loop-mediated isothermal amplification (LAMP) primer sets for the rapid detection of nusG and fadA were designed and optimized. The nusG primers yielded consistent negative results for 20 non-F. nucleatum bacterial strains, confirming the high specificity of the primers. LAMP reaction primer sensitivity was determined, and its detection rate in comparison to conventional PCR was assessed using 57 clinical stool samples. The LAMP detection limit for nusG and fadA was 22.5 and 0.225 pg µl-1, respectively, indicating that the sensitivity of this method was 10-fold higher than that of conventional PCR. These results suggest that the LAMP technique is able to effectively identify F. nucleatum via nusG as well as detect its virulence factor. To the best of our knowledge, this study is the first to report the application of LAMP for the detection of nusG and fadA in F. nucleatum. The LAMP method constitutes a sensitive and specific visual assay for the rapid detection of the pathogen F. nucleatum.
具核梭杆菌与多种人类疾病相关，如牙周病和结直肠癌（CRC）；因此，具核梭杆菌检测可能成为一种新型诊断工具。在此，我们描述了一种灵敏且快速的分子方法的开发，用于检测具核梭杆菌的两个基因：高度保守的nusG和fadA，它们编码一种关键的宿主定植因子。设计并优化了用于快速检测nusG和fadA的环介导等温扩增（LAMP）引物组。nusG引物对20种非具核梭杆菌菌株产生了一致的阴性结果，证实了引物的高特异性。测定了LAMP反应引物的灵敏度，并使用57份临床粪便样本评估了其与传统PCR相比的检测率。nusG和fadA的LAMP检测限分别为22.5和0.225 pg μl-1，表明该方法的灵敏度比传统PCR高10倍。这些结果表明，LAMP技术能够通过nusG有效鉴定具核梭杆菌，并检测其毒力因子。据我们所知，本研究是首次报道将LAMP应用于检测具核梭杆菌中的nusG和fadA。LAMP方法构成了一种灵敏且特异的可视化检测方法，用于快速检测病原体具核梭杆菌。
