梭菌属神经毒素的基因鉴定与分型

Gene identification and genotyping of clostridial neurotoxins

目的：建立梭菌属神经毒素基因快速鉴定和分型的PCR方法。方法：根据GenBank提供的肉毒毒素及破伤风毒素基因序列，综合应用多种生物学软件与在线分析平台，设计特异性及通用检测引物，对各型梭菌属毒素基因进行PCR扩增，验证扩增反应的特异性、通用性、灵敏度及重复性。结果与结论：设计了针对A，B，E，F型肉毒毒素、破伤风毒素基因轻链毒性活性区的5对特异引物，以及重链跨膜区和结合区的一对通用引物（BonAB—P01／P02，BonB-P01／P02，BonE-P01／P02，BonF-P01／P02，TET-P01／P02，TY-P01／P02）。对各型神经毒素进行特异性扩增并在型间进行交叉实验，检测特异性良好，没有交叉反应；通用扩增结果显示均得到1100bp大小的条带。目前此检测方法灵敏度可达到（1～3）×10^3个菌。该检测方法特异性强，灵敏度高，可以用于梭菌属神经毒素基因的快速筛查与鉴定分型。

Objective ：To establish a PCR method for gene identification and genotyping of elostridial neurotoxin. Methods： According to the gene sequences of botulinum and tetanus neurotoxins retrieved from the GenBank, the specific and the universal primers were designed using different softwares and on-line analytical platforms. Genes of various types of elostridial neurotoxins were specifically or universally amplified, and the specificity, sensitivity and repeatability were evaluated. Results and Conclusion：Specific primers in catalytic domain and universal primer translocation and binding domain were designed for amplifying genes encoding botulinum and tetanus neurotoxins. Their corresponding targets can be specifically amplified. As few as （ 1 - 3）×10^3 elostridial bacteria could be detected. The results demonstrated that this PCR method is specific and sensitive for the identification and genotyping of elostridial neurotoxins.