Methodological study of quantitative detection of Mycobacterium tuberculosis by immunomagnetic capture combined with PCR-ELISA / 国际检验医学杂志

ABSTRACT

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 .