摘要
目的采用免疫磁珠捕获（IMC）联合双内标PCR-ELISA（IMC-PCR-ELISA）技术,建立定量检测结核分枝杆菌的方法。方法制备能够特异性捕获结核分枝杆菌的免疫磁珠（Dynabeads）。并根据结核分枝杆菌Mtp40基因序列以及结核分枝杆菌复合体群IS6110序列,设计2对特异性引物（上游引物的5′端用生物素修饰）,以及2条与PCR扩增片段等长的内参照片段（其与扩增模板在引物区的序列相同）和3套捕获探针（3′端用地高辛标记）。先通过免疫磁珠特异性捕获结核分枝杆菌,再联合双内标PCR-ELISA技术检测结核杆菌。结果采用IMC-PCR-ELISA技术定量检测结核分枝杆菌,全过程约4h,检测限为5×10^3 copies/mL,当低内标模板浓度在30-70copies/mL,高内标模板浓度在8 000-12 000copies/mL时,计算出的浓度与实际加入的目的模板浓度之间有良好的线性关系（r^2=0.998）,未发现非特异性反应。结论成功建立了IMC-PCR-ELISA定量检测结核分枝杆菌的方法,此方法具有快速、灵敏、特异、可定量的特点。
Objective To establish a quantitative detection method for Mycobacterium tuberculosis by immunomagnetic capture combined with PCR-ELISA detection system with double internal standards（IMC-PCR-ELISA）.Methods The immunomagnetic（Dynabeads）which could specifically capture Mycobacterium tuberculosis were prepared.According to Mtp40 and IS6110gene sequence of Mycobacterium Tuberculosis,2pairs of primers（upstream primer was modified with Biotin at 5′end）,2same-length mutant fragments with PCR amplified fragments,and 3capture probes（modified with digoxigenin at 3′end）were designed.Mycobacterium tuberculosis were captured by immunomagnetic,then detected by PCR-ELISA with double internal standards.ResultsThe IMC-PCR-ELISA method could yield quantitative results in about 4hwith a detection limit at 5×10^3 copies/mL.There was a fine linear relationship between the copies of Mtp40（IS6110）in fact and in the calculation through formula when the concentrations of low internal standards were 30-70copies/mL and the concentrations of high internal standards were 8 000-12 000copies/mL（r^2=0.998）.No nonspecific amplification was observed.Conclusion A rapid and quantitative method for the detection of Mycobacterium tuberculosis was established successfully.The IMC-PCR-ELISA method was rapid,sensitive,secific and quantitative.
出处
《国际检验医学杂志》
CAS
2014年第21期2931-2933,共3页
International Journal of Laboratory Medicine
基金
镇江市社会发展基金资助项目(SH20100019)
