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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 hairpin DNA probe against Mycobacterium tuberculosis streptomycin (SM) rpsL43 codon, and try to use fluorescent spectrophotometer to directly observe the fluorescent signal after the hairpin probe in the liquid phase hybridized with the rpsL43 codon amplification product, This mutation was detected. Methods A hairpin probe targeting rpsL43 codon was designed by software Beacon designer. Fluorescence signal was detected by fluorescence spectrophotometer after hybridization between rpsL43 codon detector and probe. The sequencing results of amplification products were compared. Results Fluorescence spectrophotometer was used to detect the hybridization fluorescence signals of M. tuberculosis isolates and SM resistant rpsL43 codon amplification products. There were significant differences in fluorescence intensity between 20 SM drug-resistant groups and 10 H37RV standard strains. SM resistance The detection rate of rpsL43 codon mutation in the drug group was 70%, the mutation detection rate was 65% by sequencing, and the false-positive hairpin probe hybridization method was 1 case (504S). Conclusions The fluorescence signal of hairpin probe hybridized with fluorescence spectrophotometer is simple and sensitive. Fluorescent spectrophotometer is one of the main factors of SM resistance in Chongqing area.