CAJ | 학술논문

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M tp40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4 h ，检测限为5×103 copies/mL ，当低内标模板浓度在30～70 copies/mL ，高内标模板浓度在8000～12000 copies/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（r2＝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。
목적：채용면역자주포획（IMC）연합쌍내표PCR-ELISA（IMC-PCR-ELISA）기술，건립정량검측결핵분지간균적방법。방법제비능구특이성포획결핵분지간균적면역자주（Dynabeads?）。병근거결핵분지간균M tp40기인서렬이급결핵분지간균복합체군IS6110서렬，설계2대특이성인물（상유인물적5′단용생물소수식），이급2조여PCR확증편단등장적내삼조편단（기여확증모판재인물구적서렬상동）화3투포획탐침（3′단용지고신표기）。선통과면역자주특이성포획결핵분지간균，재연합쌍내표PCR-ELISA기술검측결핵간균。결과채용IMC-PCR-ELISA기술정량검측결핵분지간균，전과정약4 h ，검측한위5×103 copies/mL ，당저내표모판농도재30～70 copies/mL ，고내표모판농도재8000～12000 copies/mL시，계산출적농도여실제가입적목적모판농도지간유량호적선성관계（r2＝0．998），미발현비특이성반응。결론성공건립료IMC-PCR-ELISA정량검측결핵분지간균적방법，차방법구유쾌속、령민、특이、가정량적특점。
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 IS6110 gene sequence of Mycobacterium Tuberculosis ,2 pairs of primers(upstream primer was modified with Biotin at 5′end) ,2 same-length mutant fragments with PCR amplified fragments ,and 3 capture probes(modified with digoxigenin at 3′end) were designed .Myco-bacterium tuberculosis were captured by immunomagnetic ,then detected by PCR-ELISA with double internal standards .Results The IMC-PCR-ELISA method could yield quantitative results in about 4 h with a detection limit at 5 × 103 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 30-70 copies/mL and the concentrations of high internal standards were 8 000-12 000 copies/mL (r2 =0 .998) .No nonspecific amplification was observed .Conclusion A rapid and quantitative method for the detection of Myco-bacterium tuberculosis was established successfully .The IMC-PCR-ELISA method was rapid ,sensitive ,secific and quantitative .