运用荧光分光光度仪观测SM耐药rpsL43密码子发.docVIP

运用荧光分光光度仪观测SM耐药rpsL 43密码子发夹探针液相杂交信号 陈庆海1，张 波1，黄君富1，华 兴1，张 雪1，匡 红2，王云霞1，府伟灵1( （1第三军医大学西南医院检验科，重庆400038；2解放军第452医院，成都 610021） 摘 要：目的 设计针对结核杆菌耐链霉素(Sm) rpsL 43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rpsL 43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设计标准株 hybridisation fluorescence of rpsL 43 codon of SM resistant in liquid by hair clamp probe technology with Fluorescence spectrophotometer CHEN Qin-hai1，ZHANG Bo1，HUANG Jun-fu1，HUA Xing1，ZHANG Xue1，KUANG Hong2，WANG Yun-xia1，FU Wei-ling1 (1The clinical lab of Southwest Hospital，the Third Military Medical University，Chongqing 400038，2The 452 hospital of PLA，Chengdu，610021，China) Abstract: Objective To design hair clamp DNA probe of rpsL 43 Codon of SM resistant of MTB. To try to detecting hybridisation fluorescence between the amplified product of rpsL 43 codon and hair clamp probe in liquid by Fluorescence spectrophotometer to detect mutation site. Methods The software，Beacon designer，was used to design the hair clamp probe of rpsL 43 Codon and detecting fluorescence signal of hybridization between the amplified product and probe，and compare between the way and sequencing of the amplification products. Results The difference between PCR products from standard strain and rpsL 43 Codon of SM resistant one is obvious in detecting the hybridization fluorescent light by Fluorescence spectrophotometer. We detected fluorescent light signal between the 20 SM resistant strains and 10 H37RV standard strains. The mutation rate of SM resistant detected is about 70%，and the rate of sequencing is about 65%. The 1 strain (504S) false positive is found in hair clamp probe way. Conclusion Fluorescence spectrophotometer have characteristics such as high sensitiveness，simple to directly detect the hybridization fluorescent light of hair clamp probe in liquid. The mutation site of RpsL 43 Codon is one of the main reasons of SM resistant MTB in Chongqing. Key words: Hair clamp probe；RpsL 43 Codon of SM resistant；Fluorescence spectr