基于TaqMan的定量PCR技术的开发，用于灵敏且选择性地检测人粪便中产毒素艰难梭菌。
Development of TaqMan-based quantitative PCR for sensitive and selective detection of toxigenic Clostridium difficile in human stools.
作者信息
Kubota Hiroyuki, Sakai Takafumi, Gawad Agata, Makino Hiroshi, Akiyama Takuya, Ishikawa Eiji, Oishi Kenji
机构信息
Yakult Honsha European Research Center for Microbiology ESV, Gent-Zwijnaarde, Belgium; Yakult Central Institute, Tokyo, Japan.
Yakult Central Institute, Tokyo, Japan.
出版信息
PLoS One. 2014 Oct 31;9(10):e111684. doi: 10.1371/journal.pone.0111684. eCollection 2014.
BACKGROUND
Clostridium difficile is the main cause of nosocomial diarrhea, but is also found in asymptomatic subjects that are potentially involved in transmission of C. difficile infection. A sensitive and accurate detection method of C. difficile, especially toxigenic strains is indispensable for the epidemiological investigation.
METHODS
TaqMan-based quantitative-PCR (qPCR) method for targeting 16S rRNA, tcdB, and tcdA genes of C. difficile was developed. The detection limit and accuracy of qPCR were evaluated by analyzing stool samples spiked with known amounts of C. difficile. A total of 235 stool specimens collected from 82 elderly nursing home residents were examined by qPCR, and the validity was evaluated by comparing the detection result with that by C. difficile selective culture (CDSC).
RESULTS
The analysis of C. difficile-spiked stools confirmed that qPCR quantified whole C. difficile (TcdA+TcdB+, TcdA-TcdB+, and TcdA-TcdB- types), TcdB-producing strains (TcdA+TcdB+ and TcdA-TcdB+ types), and TcdA-producing strains (TcdA+TcdB+ type), respectively, with a lower detection limit of 103 cells/g of stool. Of the 235 specimens examined, 12 specimens (5.1%) were C. difficile-positive by qPCR: TcdA+TcdB+ strain in six specimens and TcdA-TcdB- strain in the other six. CDSC detected C. difficile in 9 of the 12 specimens, and toxigenic types of the isolates from the 9 specimens were consistent with those identified by qPCR, supporting the validity of our qPCR method. Moreover, the qPCR examination revealed that the carriage rate of whole C. difficile and that of toxigenic strains in the 82 subjects over a 6-month period ranged from 2.4 to 6.8% and 1.2 to 3.8%, respectively. An average qPCR count of C. difficile detected was 104.5 cells/g of stool, suggesting that C. difficile constituted a very small fraction of intestinal microbiota.
CONCLUSION
Our qPCR method should be an effective tool for both clinical diagnosis and epidemiological investigation of C. difficile.
背景
艰难梭菌是医院获得性腹泻的主要病因，但在无症状个体中也有发现，这些个体可能参与艰难梭菌感染的传播。对于艰难梭菌，尤其是产毒菌株，一种灵敏且准确的检测方法对于流行病学调查而言必不可少。
方法
开发了基于TaqMan的定量PCR（qPCR）方法，用于靶向艰难梭菌的16S rRNA、tcdB和tcdA基因。通过分析添加了已知量艰难梭菌的粪便样本，评估qPCR的检测限和准确性。对从82名老年疗养院居民收集的235份粪便标本进行qPCR检测，并通过将检测结果与艰难梭菌选择性培养（CDSC）结果进行比较来评估其有效性。
结果
对添加了艰难梭菌的粪便进行分析证实，qPCR分别对完整的艰难梭菌（TcdA+TcdB+、TcdA-TcdB+和TcdA-TcdB-型）、产TcdB菌株（TcdA+TcdB+和TcdA-TcdB+型）和产TcdA菌株（TcdA+TcdB+型）进行定量，粪便中检测限低至103个细胞/g。在检测的235份标本中，12份标本（5.1%）通过qPCR检测为艰难梭菌阳性：6份标本为TcdA+TcdB+菌株，另外6份为TcdA-TcdB-菌株。CDSC在12份标本中的9份中检测到艰难梭菌，9份标本分离株的产毒类型与qPCR鉴定的一致，支持了我们qPCR方法的有效性。此外，qPCR检测显示，82名受试者在6个月期间完整艰难梭菌的携带率和产毒菌株的携带率分别为2.4%至6.8%和1.2%至3.8%。检测到的艰难梭菌平均qPCR计数为104.5个细胞/g粪便，表明艰难梭菌在肠道微生物群中所占比例非常小。
结论
我们的qPCR方法应是艰难梭菌临床诊断和流行病学调查的有效工具。
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