运用荧光分光光度仪观测结核杆菌耐链霉素耐药rpsL 43密码子发夹探针液相杂交信号  

Liquid hybridization assay for detecting rpsL 43 of streptomycin-resistant Mycobacterium tuberculosis with fluorescence spectrophotometer

作　　者：陈庆海[1] 华兴[1] 张波[1] 张雪[1] 黄君富[1] 匡红 府伟灵[1] 

机构地区：[1]第三军医大学西南医院检验科,重庆400038 [2]解放军等452医院检验科,成都610021

出　　处：《第三军医大学学报》2009年第24期2421-2424,共4页Journal of Third Military Medical University

基　　金：国家自然科学基金(30970879, 30571775);重庆市自然科学基金(CSTC 2009BB5140);重庆市计生委课题(2008-5)~~

摘　　要：目的设计针对结核杆菌耐链霉素(SM)rpsL43密码子的发夹型DNA探针,尝试运用荧光分光光度仪直接观测液相中发夹探针与rpsL43密码子扩增产物杂交后荧光信号,从而检出该位点突变。方法运用软件Beacon designer设计针对rpsL43密码子的发夹探针,应用荧光分光光度仪检测rpsL43密码子扩增片段与探针杂交后荧光信号,并比较扩增产物测序结果。结果通过荧光分光光度仪观测到结核杆菌标准株及SM耐药rpsL43密码子扩增产物杂交荧光信号存在显著差异;20株SM耐药组与10株H37RV标准株对照组荧光信号强度比较,SM耐药组rpsL43密码子突变检出率为70%,测序法突变检出率为65%,发夹探针杂交法假阳性株1例(504S)。结论应用荧光分光光度仪直接观测发夹探针液相杂交荧光信号具有简单、灵敏等优特点;rpsL43密码子点突变是重庆地区SM耐药的主要因素之一。Objective To design a hair clamp DNA probe of rpsL 43 from streptomycin （SM） -resistant Mycobacterium tuberculosis and detect the liquid hybridization fluorescence signal between the amplified product of rpsL 43 and our hair clamp probe by fluorescence spectrophotometry in order to detect whether there is muta- tion at the site. Methods The software, Beacon designer, was used to design the hair clamp probe of rpsL 43. Fluorescence signal of hybridization between the amplified products of rpsL 43 from 20 strains isolated from Chongqing region and our probe was detected with fluorescence spectrophotometry. The sequences of the amplification products from different strains were compared. Results The difference in rpsL 43 PCR products between standard strain and SM-resistant one was very obvious when detecting the hybridization fluorescent signal by fluorescence spectrophotometer. We found fluorescent signal between our probe and the rpsL 43amplified products from the 20 SM-resistant strains and 10 H37RV standard strains. The mutation rate of SM-resistant strains was about 70% by our liquid hybridization assay, while the rate by sequencing was about 65%. Our assay found a false positive strain （504S）. Conclusion Our liquid hybridization assay is easy and sensitive to detect rpsL 43 of SM-resistant Mycobacterium tuberculosis. The mutation site of RpsL 43 is one of the main rea- sons to cause SM-resistance in Mycobacterium tuberculosis in Chongqing region.

关 键 词：发夹型DNA探针 SM耐药rpsL 43密码子 荧光分光光度仪 液相杂交 

分 类 号：R378.911[医药卫生—病原生物学] R394.3[医药卫生—基础医学]