艰难梭菌数字化聚合酶链反应-核糖体分型方法及参考图谱的建立

Establishment of digital polymerase chain reaction-ribotyping and database for Clostridium difficile genotyping

目的建立艰难梭菌数字化PCR-核糖体分型方法及常见基因型的参考图谱。方法利用荧光毛细管电泳技术代替琼脂糖凝胶电泳检测艰难梭菌PCR-核糖体分型产物，建立数字化PCR-核糖体分型方法；利用40株分属于10种核糖体型（RT）的艰难梭菌参考菌株构建常见基因型的数字化参考图谱。结果荧光毛细管电泳技术准确地检测出40株艰难梭菌参考菌株的PCR-核糖体分型片段，并以数字化的形式呈现，10种核糖体型艰难梭菌的PCR-核糖体分型数字化图谱差异明显。同为RT027的21株菌株间、RT002的3株菌株间和RT015的2株菌株间的数字化分型结果相似性都非常高，而7株RT001菌株则可分为4种亚型，2株RT014分为2种亚型。结论成功建立艰难梭菌数字化PCR-核糖体分型方法，同时构建了艰难梭菌10种核糖体型的数字化PCR-核糖体分型参考数据库。

Objective To develop a digital polymerase chain reaction （PCR）-rihotyping method and database for Clostridiurn difficile genotyping. Methods Sequencer based fluorescence capillary gel electrophoresis was used, instead of agarose gel electrophoresis, to establish the digital PCR-ribotyping of Clostridium difficile. Forty Clostridium difficile reference strains, consisting of 10 PCR-ribotypes （RT）, were genotyped by the new digital PCR-rihotyping method to set-up the database. Results The sequencer based fluorescence capillary gel electrophoresis correctly detected PCR-ribotyping products of the 40 reference strains, and showed as digital figure; significant differences of these digital figures were found between the 10 RT. High similar digital figures were shown in twenty-one RT027 strains, three RT002 strains and two RT014 strains. However, seven RT001 strains were typed as four subtypes, and two RT014 strains as two subtypes, respectively. Conclusion A digital PCR-ribotyping, and a reference database consisting of 10 RT are successfully established.