CAJ | 학술논문

目的建立免疫磁珠荧光量子点技术检测MTB的方法,探讨最佳检测条件并进行初步评估.方法以MTB标准株H37 Rv、12种非结核分枝杆菌(NTM)标准株及3种非分枝杆菌为检测对象,免疫磁珠和功能化荧光量子点作用为对照组.利用湿化学方法制备纳米磁珠和荧光量子点,分别与MTB结合肽H8偶联,获得同时与H37 Rv作用的免疫磁珠和功能化荧光量子点,形成三元复合物结构,通过测量荧光值及荧光显微镜观察判定样品中是否存在MTB,建立纳米技术检测MTB的新方法,进一步探讨免疫磁珠和功能化荧光量子点的最佳检测浓度及作用时间.利用菌悬液和模拟痰标本,对该检测方法的检测下限和特异度进行初步评估.结果偶联的免疫磁珠和功能化荧光量子点均能与H37 Rv结合,镜下可见其与H37 Rv作用的三元复合物,实验组与对照组的荧光值比值约为4:1；免疫磁珠和功能化荧光量子点最佳检测浓度均为100 mg/L,最佳作用时间为2 h;H37Rv菌悬液和模拟痰标本检测下限均为103 CFU/ml；特异度检测结果,3种非分枝杆菌在显微镜下均未见红色荧光,荧光值小于对照组,为阴性；12种NTM菌悬液和模拟痰标本检测时,仅副偶发分枝杆菌、金色分枝杆菌、耻垢分枝杆菌和偶发分枝杆菌在显微镜下可见红色荧光,荧光值与对照组的比值均＞2,判断为阳性,其余均为阴性.结论免疫磁珠荧光量子点技术检测技术有可能成为检测MTB的新方法,但其临床应用价值尚有待进一步评估.
목적 건립면역자주형광양자점기술검측MTB적방법,탐토최가검측조건병진행초보평고.방법 이MTB표준주H37 Rv、12충비결핵분지간균(NTM)표준주급3충비분지간균위검측대상,면역자주화공능화형광양자점작용위대조조.이용습화학방법제비납미자주화형광양자점,분별여MTB결합태H8우련,획득동시여H37 Rv작용적면역자주화공능화형광양자점,형성삼원복합물결구,통과측량형광치급형광현미경관찰판정양품중시부존재MTB,건립납미기술검측MTB적신방법,진일보탐토면역자주화공능화형광양자점적최가검측농도급작용시간.이용균현액화모의담표본,대해검측방법적검측하한화특이도진행초보평고.결과 우련적면역자주화공능화형광양자점균능여H37 Rv결합,경하가견기여H37 Rv작용적삼원복합물,실험조여대조조적형광치비치약위4:1；면역자주화공능화형광양자점최가검측농도균위100 mg/L,최가작용시간위2 h;H37Rv균현액화모의담표본검측하한균위103 CFU/ml；특이도검측결과,3충비분지간균재현미경하균미견홍색형광,형광치소우대조조,위음성；12충NTM균현액화모의담표본검측시,부부우발분지간균、금색분지간균、치구분지간균화우발분지간균재현미경하가견홍색형광,형광치여대조조적비치균＞2,판단위양성,기여균위음성.결론 면역자주형광양자점기술검측기술유가능성위검측MTB적신방법,단기림상응용개치상유대진일보평고.
Objective To establish a detection method for Mycobacterium tuberculosis (MTB) by immunomagnetic beads combined with functionalized fluorescent quantum dots technology,and to investigate the optimal test condition and the diagnostic value of this method.Methods MTB standard strain H37 Rv was used as detection object.Nanobeads and quantum dots were prepared by using wet chemical method,and conjugated separately with MTB binding peptide H8 to obtain immunomagnetic beads and functionalized fluorescent quantum dots,which could react with H37Rv simultaneously and form a ternary complex structure.Based on measurement of the fluorescence value and observation under fluorescence microscopy to determine if MTB existed in the sample,a new detection method of MTB using nanotechnology was established.The optimal detection concentration and reaction time of immunomagnetic beads and quantum dots were investigated,and the detection limit and specificity of this detection method were evaluated by using bacterial suspension and simulation sputum samples.Results By fluorescence microscopy examination,it was found that conjugated immunomagnetic beads and functionalized fluorescent quantum dots both bound with H37 Rv and formed the ternary complex structure.The fluorescent value ratio of the experimental group and the control group could be 4: 1.The best detection concentration of immunomagnetic beads and functionalized fluorescent quantum dots was 100 mg/L and the optimal incubation time was 2 h.The detection limit of H37Rv bacterial suspension and simulation sputum sample were both 103 CFU/ml.The detection results for 3 non-mycobacteria were all negative,while for the 12 types of NTM,only Mycobacterium parafortuitum,Mycobacterium aurum,Mycobacterium smegmatis and Mycobacterium fortuitum were positive,and others were all negative.Conclusion The detection method of immunomagnetic beads combined with fluorescence quantum dots can be a new detection method for MTB,but the clinical value needs to be evaluated further.