College of Public Health, Division of Environmental Health Sciences, The Ohio State University, Columbus, Ohio 43210, USA.
J Microbiol Methods. 2010 Sep;82(3):311-8. doi: 10.1016/j.mimet.2010.07.012. Epub 2010 Jul 17.
A rapid and specific gyrB-based real-time PCR system has been developed for detecting Bacteroides fragilis as a human-specific marker of fecal contamination. Its specificity and sensitivity was evaluated by comparison with other 16S rRNA gene-based primers using closely related Bacteroides and Prevotella. Many studies have used 16S rRNA gene-based method targeting Bacteroides because this genus is relatively abundant in human feces and is useful for microbial source tracking. However, 16S rRNA gene-based primers are evolutionarily too conserved among taxa to discriminate between human-specific species of Bacteroides and other closely related genera, such as Prevotella. Recently, one of the housekeeping genes, gyrB, has been used as an alternative target in multilocus sequence analysis (MLSA) to provide greater phylogenetic resolution. In this study, a new B. fragilis-specific primer set (Bf904F/Bf958R) was designed by alignments of 322 gyrB genes and was compared with the performance of the 16S rRNA gene-based primers in the presence of B. fragilis, Bacteroides ovatus and Prevotella melaninogenica. Amplicons were sequenced and a phylogenetic tree was constructed to confirm the specificity of the primers to B. fragilis. The gyrB-based primers successfully discriminated B. fragilis from B. ovatus and P. melaninogenica. Real-time PCR results showed that the gyrB primer set had a comparable sensitivity in the detection of B. fragilis when compared with the 16S rRNA primer set. The host-specificity of our gyrB-based primer set was validated with human, pig, cow, and dog fecal samples. The gyrB primer system had superior human-specificity. The gyrB-based system can rapidly detect human-specific fecal source and can be used for improved source tracking of human contamination.
一种快速且特异的基于 gyrB 的实时 PCR 系统已被开发出来，用于检测脆弱拟杆菌，作为粪便污染的人源特异性标志物。通过与其他基于 16S rRNA 基因的引物比较，评估了其特异性和敏感性，这些引物针对的是密切相关的拟杆菌属和普雷沃氏菌属。许多研究使用基于 16S rRNA 基因的方法来靶向拟杆菌属，因为该属在人类粪便中相对丰富，并且可用于微生物溯源。然而，基于 16S rRNA 基因的引物在分类上的进化过于保守，无法区分人源特异性的拟杆菌属物种和其他密切相关的属，如普雷沃氏菌属。最近，一种新的管家基因 gyrB 已被用作多位点序列分析（MLSA）中的替代靶标，以提供更高的系统发育分辨率。在本研究中，通过对 322 个 gyrB 基因的比对，设计了一套新的脆弱拟杆菌属特异性引物（Bf904F/Bf958R），并与 16S rRNA 基因引物在存在脆弱拟杆菌、卵形拟杆菌和黑色素普雷沃氏菌时的性能进行了比较。对扩增子进行测序，并构建了系统发育树，以确认引物对脆弱拟杆菌的特异性。基于 gyrB 的引物成功地区分了脆弱拟杆菌与卵形拟杆菌和黑色素普雷沃氏菌。实时 PCR 结果表明，与 16S rRNA 引物相比，gyrB 引物在脆弱拟杆菌检测中的灵敏度相当。用人类、猪、牛和狗粪便样本验证了我们基于 gyrB 的引物的宿主特异性。gyrB 引物具有优越的人源特异性。基于 gyrB 的系统可快速检测人源特异性粪便来源，可用于改进人源污染的溯源。
