Kato H, Kato N, Watanabe K, Iwai N, Nakamura H, Yamamoto T, Suzuki K, Kim S M, Chong Y, Wasito E B
Institute of Anaerobic Bacteriology, Gifu University School of Medicine, Japan.
J Clin Microbiol. 1998 Aug;36(8):2178-82. doi: 10.1128/JCM.36.8.2178-2182.1998.
Toxigenic strains of Clostridium difficile have been reported to produce both toxins A and B nearly always, and nontoxigenic strains have been reported to produce neither of these toxins. Recent studies indicate that it is not always true. We established a PCR assay to differentiate toxin A-negative, toxin B-positive (toxin A-, toxin B+) strains from both toxin-positive (toxin A+, toxin B+) strains and both toxin-negative (toxin A-, toxin B-) strains as an alternative to cell culture assay and enzyme-linked immunosorbent assay (ELISA). By using the PCR primer set NK11 and NK9 derived from the repeating sequences of the toxin A gene, a shorter segment (ca. 700 bp) was amplified from toxin A-, toxin B+ strains compared to the size of the segment amplified from toxin A+, toxin B+ strains (ca. 1,200 bp), and no product was amplified from toxin A-, toxin B-strains. We examined a total of 421 C. difficile isolates by PCR. Of these, 48 strains showed a shorter segment by the PCR, were negative by ELISAs for the detection of toxin A, and were positive by cell culture assay. Although the cytotoxin produced by the toxin A-, toxin B+ strains was neutralized by anti-toxin B serum, the appearance of the cytotoxic effects on Vero cell monolayers was distinguishable from that of toxin A+, toxin B+ strains. By immunoblotting, the 44 toxin A-, toxin B+ strains were typed to serogroup F and the remaining four strains were serogroup X. Pulsed-field gel electrophoresis separated the 48 strains into 19 types. The PCR assay for the detection of the repeating sequences combined with PCR amplification of the nonrepeating sequences of either the toxin A or the toxin B gene is indicated to be useful for differentiating toxin A-, toxin B+ strains from toxin A+, toxin B+ and toxin A-, toxin B- strains and will contribute to elucidation of the precise role of toxin A-, toxin B+ strains in intestinal diseases.
据报道，艰难梭菌的产毒菌株几乎总是同时产生毒素A和毒素B，而非产毒菌株则不产生这两种毒素。然而，最近的研究表明情况并非总是如此。我们建立了一种聚合酶链反应（PCR）检测方法，以区分毒素A阴性、毒素B阳性（毒素A -，毒素B +）菌株与毒素阳性（毒素A +，毒素B +）菌株以及毒素阴性（毒素A -，毒素B -）菌株，作为细胞培养检测和酶联免疫吸附测定（ELISA）的替代方法。通过使用源自毒素A基因重复序列的PCR引物对NK11和NK9，与从毒素A +，毒素B +菌株扩增的片段大小（约1200 bp）相比，从毒素A -，毒素B +菌株扩增出的片段较短（约700 bp），并且从毒素A -，毒素B -菌株未扩增出产物。我们通过PCR共检测了421株艰难梭菌分离株。其中，48株通过PCR显示出较短的片段，ELISA检测毒素A为阴性，细胞培养检测为阳性。尽管毒素A -，毒素B +菌株产生的细胞毒素被抗毒素B血清中和，但对Vero细胞单层的细胞毒性作用的表现与毒素A +，毒素B +菌株不同。通过免疫印迹法，44株毒素A -，毒素B +菌株被鉴定为F血清型，其余4株为X血清型。脉冲场凝胶电泳将这48株菌株分为19种类型。检测重复序列的PCR检测方法与毒素A或毒素B基因非重复序列的PCR扩增相结合，被证明可用于区分毒素A -，毒素B +菌株与毒素A +，毒素B +以及毒素A -，毒素B -菌株，这将有助于阐明毒素A -，毒素B +菌株在肠道疾病中的精确作用。
