麻风杆菌PCR检测方法的优化及应用  

Optimization and Application of PCR Detection for Mycobacterium Leprae

作　　者：叶理[1] 吕成志[1] 靳亚丽[1] 王会翔[1] 张振国[1] 宋顺鹏[1] 

机构地区：[1]大连市皮肤病医院,辽宁大连116021

出　　处：《皮肤性病诊疗学杂志》2016年第1期23-26,共4页Journal of Diagnosis and Therapy on Dermato-venereology

摘　　要：目的:研建一种适用于临床中检测麻风杆菌(ML)的分子生物学方法,并优化检测条件以提高检测方法的灵敏度。方法:用麻风杆菌纯DNA作模板,对PCR反应体系中模板浓度、引物浓度等反应条件进行优化,以建立高敏感性的PCR反应体系,并与实时荧光定量PCR(Real-time PCR)方法比较,对麻风病人标本进行检测,确定优化后两法的反应体系及两法的临床检测意义。结果:经过大量的实验比对表明,反应体系引物含量为0.2μL×100μmol时,模板浓度10-1条ML/m L可以作为稳定的可检测到条带的模板浓度的下限。对52例各型麻风患者标本的检测结果显示常规PCR检测阳性数(49/52)略高于Real-time PCR(47/52),但Real-time PCR操作程序简单和需时短,且成本低。结论:通过对PCR反应条件中DNA模板浓度和引物浓度的优选,提高了PCR检测麻风杆菌DNA的灵敏度;对于不同PCR方法的选择方面,Real-time PCR比常规PCR简捷快速,而常规PCR灵敏度略高于Realtime PCR。Objective： To establish a molecular biological method with high sensitivity for detecting Mycobacterium leprae（ ML） in clinics. Methods： Establish a highly sensitive PCR reaction system by optimizing the reaction conditions of PCR reaction system using the pure DNA of Mycobacterium leprae and compare the efficacy and significance of two systems-conventional PCR（ c PCR） and real-time PCR as clinical detection methods. Results： The results indicated that 10- 1ML / m L is the lowest DNA concentration which can be steadily detected when the primer content is0. 2 μL × 100 μmol / L. The number of positive results of c PCR（ 49 /52） was slightly higher than that of Real-time PCR（ 47 /52） according to the results detected from the samples of leprosy patients. Conclusion： Through the optimization of reaction conditions in the PCR reaction,such as DNA template concentration and primer concentration,the sensitivity of Mycobacterium leprae DNA detection can be improved. The c PCR and Real-time PCR were both highly sensitive methods. However,Real-time PCR detection is more simple and rapid than c PCR,while c PCR is slightly more accurate and sensitive compare to Real-time PCR.

关 键 词：麻风分枝杆菌 聚合酶链反应 实时定量聚合酶链反应 

分 类 号：R755[医药卫生—皮肤病学与性病学]