IDENTIFICATION OF TOXIGENIC CLOSTRIDIUM-DIFFICILE BY THE POLYMERASE CHAIN-REACTION
IDENTIFICATION OF TOXIGENIC CLOSTRIDIUM-DIFFICILE BY THE POLYMERASE CHAIN-REACTION
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DOI：
10.1128/jcm.29.1.33-37.1991
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发表时间：
1991-01-01
影响因子：
9.4
通讯作者：
UENO, K
中科院分区：
文献类型：
--
作者：
KATO, N;OU, CY;UENO, K
艰难梭菌的产毒素菌株是伪膜性结肠炎和抗微生物剂相关性腹泻和结肠炎的病原体。 通过使用高度敏感的细胞培养物常规测定促细胞增殖性。 我们用一种简单、快速的聚合酶链反应（PCR）方法来区分致突变株和非致突变株。很难 两套寡核苷酸引物对来自毒素A基因的非重复序列被用来扩增546-和252-bp的DNA片段。 使用来自毒素A基因的重复序列的引物对来扩增1，266-bp的DNA产物。 扩增产物通过聚丙烯酰胺凝胶电泳，然后溴化乙锭染色进行可视化。 35株细胞毒C.检测的艰难梭菌产生预期的扩增DNA。 相比之下，检测的26种非细胞毒性菌株均未得到阳性结果。 虽然C.艰难梭菌与索氏梭菌毒素有血清学交叉反应，我们在两株细胞毒性菌株和七株非细胞毒性菌株中未检测到任何扩增的DNA。sordellii。 其他20种梭菌的30株PCR均为阴性。 用亚基因组探针对HindIII酶切后的基因组DNA进行Southern杂交，结果表明，在转基因菌株中只有一条杂交带，而在非转基因菌株中没有。 PCR是一种敏感、特异的快速鉴定产毒C.很难 非转基因C. difficile似乎缺乏C.艰难梭菌毒素A基因。
Toxigenic strains of Clostridium difficile are causative agents of pseudomembranous colitis and antimicrobial agent-associated diarrhea and colitis. The toxigenicity is routinely assayed by using highly sensitive cell cultures. We used a simple and rapid polymerase chain reaction (PCR) assay to differentiate toxigenic and nontoxigenic strains of C. difficile. Two sets of oligonucleotide primer pairs derived from nonrepeating sequences of the toxin A gene were used to amplify 546- and 252-bp DNA fragments. A primer pair derived from repeating sequences of the toxin A gene was used to amplify a 1,266-bp DNA product. Amplified products were visualized by polyacrylamide gel electrophoresis followed by ethidium bromide staining. All 35 cytotoxic strains of C. difficile tested generated the expected amplified DNA. In contrast, none of the 26 noncytotoxic strains tested gave positive results. Although the toxins of C. difficile have been demonstrated to cross-react serologically with the toxins of Clostridium sordellii, we did not detect any amplified DNA in two cytotoxic strains or seven noncytotoxic strains of C. sordellii. PCR was negative in all 30 strains of 20 other Clostridium species. Southern hybridization of Hindlll-digested genomic DNA by use of subgenomic probes showed a single hybridization band in toxigenic strains but not in nontoxigenic strains. PCR appears to be a sensitive and specific assay for the rapid identification of toxigenic C. difficile. Nontoxigenic C. difficile appeared to lack the C. difficile toxin A gene.