Quantification ofMycobacterium tuberculosis DNA using PCR and a simple dot blot-based DNA enzyme assay (DEA)

Conclusions Use of PCR for quantification of microorganisms has certain limitations. Methods currently under investigation, such as covalent binding of modified DNA onto the surface of microwells or coupling to magnetic beads with the aid of biotin-avidin, are hampered by problems concerning immobilization of DNA on a solid phase. Moreover, these strategies are laborious, including several washing steps and complicated detection systems (sandwich hybridization).We have developed an alternative method, exploiting the reliability of covalent DNA binding to positively charged nylon membranes enabling easy to handle direct hybridization. The method is adaptable for routine use in clinical laboratories. We have shown results of a quantitative assay to measure bacterial load of MTB. Quantification of MTB may have value in: (i) monitoring patients under anti-myobacterial therapy, and (ii) early in vitro drug susceptibility testing.