Sloan Lynne M, Duresko Brian J, Gustafson Daniel R, Rosenblatt Jon E
Division of Clinical Microbiology, Mayo Clinic, Rochester, Minnesota 55906, USA.
J Clin Microbiol. 2008 Jun;46(6):1996-2001. doi: 10.1128/JCM.00032-08. Epub 2008 Apr 23.
We have developed a rapid real-time PCR method using fluorescence resonance energy transfer probes and the LightCycler (Roche Diagnostics), which will detect the presence of the tcdC gene of Clostridium difficile in stool samples. Our PCR method also will identify the presence of base pair deletions, one of which (18 bp) has been associated with the "epidemic" toxin-hyperproducing strains. We compared the results of this PCR with those of three C. difficile toxin-detecting enzyme immunoassays (EIAs), an EIA for the detection of glutamate dehydrogenase (GDH), and culture of C. difficile. A total of 200 stool specimens were studied by the methods under comparison. C. difficile was isolated from 49 specimens by culture, and 44 of these were confirmed as containing one of the genes associated with toxin production ("toxigenic culture"). Using toxigenic culture as the "gold standard", the sensitivities, specificities, and positive and negative predictive values, respectively, of the assays were 48%, 98%, 88%, and 87% for the Premier toxin A and B test; 48%, 99%, 91%, and 87% for the ImmunoCard toxin A & B test; 48%, 84%, 46%, and 85% for the Xpect C. difficile toxin A/B test; 32%, 100%, 100%, and 84% for the Triage C. difficile panel (for toxin A); and 86%, 97%, 90%, and 96% for the LightCycler PCR. Thus, in comparison to the sensitivity of toxigenic culture, the sensitivities of the toxin immunoassays were unacceptably low, while the LightCycler real-time PCR assay for the detection of the tcdC gene of C. difficile is sensitive and specific.
我们开发了一种使用荧光共振能量转移探针和罗氏诊断公司的LightCycler的快速实时PCR方法，该方法可检测粪便样本中艰难梭菌tcdC基因的存在。我们的PCR方法还能识别碱基对缺失的情况，其中一种（18bp）与“流行”的毒素高产菌株有关。我们将该PCR结果与三种艰难梭菌毒素检测酶免疫测定法（EIA）、一种检测谷氨酸脱氢酶（GDH）的EIA以及艰难梭菌培养结果进行了比较。总共200份粪便标本通过这些比较方法进行了研究。通过培养从49份标本中分离出艰难梭菌，其中44份被确认为含有与毒素产生相关的基因之一（“产毒培养”）。以产毒培养作为“金标准”，Premier毒素A和B检测的灵敏度、特异性、阳性和阴性预测值分别为48%、98%、88%和87%；ImmunoCard毒素A和B检测为48%、99%、91%和87%；Xpect艰难梭菌毒素A/B检测为48%、84%、46%和85%；Triage艰难梭菌检测板（针对毒素A）为32%、100%、100%和84%；LightCycler PCR为86%、97%、90%和96%。因此，与产毒培养的灵敏度相比，毒素免疫测定法的灵敏度低得令人无法接受，而用于检测艰难梭菌tcdC基因的LightCycler实时PCR测定法灵敏且特异。
