食品中A型肉毒梭菌PCR-DHPLC检测方法的建立

Denaturing high performance liquid chromatography (DHPLC)used in the detection of Clostridium botulinum-A

目的:建立食品中A型肉毒梭菌的快速检测方法。方法:应用PCR结合变性高效液相色谱(DHPLC)技术,以A型肉毒梭菌的A型肉毒神经毒素基因作为靶基因设计特异性引物,PCR扩增的产物经DHPLC技术进行快速检测。以非A型肉毒梭菌,产气荚膜梭菌等23株参考菌株做特异性实验;A型肉毒梭菌DNA稀释成不同梯度,做灵敏度实验。结果:与传统的检测方法相比,该方法具有很好的特异性,方法灵敏度较高,最低检出限可达到为111ng/tube。结论:该方法可以快速、准确检测A型肉毒梭菌,是食品中致病菌快速检测的新技术。

Objective：Quick checking method of Clostridium botulinum was established by polymerase chain reaction（PCR）and denaturing high-performance liquid chromatography（DHPLC）.Methods：With gene of Clostridium botulinum＇ type A botulinum neurotoxin as target gene,the primer was designed and PCR system was optimized.And the twenty-three test strains such as Clostridium perfringens were tested by specific detection.Then different grades of standard strain were obtained by diluting and sensitivity was detected.Results：The PCR-DHPLC methods could test Clostridium botulinum exclusively and sensitively which with the lowest amount of detecting was111ng/tube.Conclusion：The PCR-DHPLC method was rapid and accurate in detection of Clostridium botulinum in food.