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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1 100 bp大小的条带。目前此检测方法灵敏度可达到(1~3)×103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 rapid identification and typing of Clostridium neurotoxin genes. Methods: According to the sequences of botulinum toxin and tetanus toxin genes provided by GenBank, a variety of biological softwares and online analysis platforms were used to design specific and universal detection primers to amplify and verify the Clostridium toxin genes Amplification reaction specificity, versatility, sensitivity and repeatability. RESULTS AND CONCLUSION: Five pairs of primers specific to the botulinum toxin A, B, E, F genotoxic regions of tetanus toxin gene and a pair of universal primers of the transmembrane region and binding region of heavy chain (BonAB -P01 / P02, BonB-P01 / P02, BonE-P01 / P02, BonF-P01 / P02, TET-P01 / P02, TY-P01 / P02). All types of neurotoxins were specifically amplified and cross-tested in the type. The specificity was good and there was no cross-reaction. The common amplification results showed that each band had a size of 1 100 bp. At present, the sensitivity of this test method can reach (1 ~ 3) × 103 bacteria. The method has high specificity and high sensitivity and can be used for the rapid screening and identification of Clostridium neurotoxin genes.