New developments in molecular diagnostics

Abstract

Molecular diagnostic methods offer new opportunities in the diagnosis of infectious diseases. Compared to immunological procedures they provide excellent sensitivities and specificities. Especially with respect to the safety of blood products, the PCR-technology is well suited. Basically molecular diagnostic methods can be separated into specimen preparation and amplification/detection. As to specimen preparation a new technology has been developed based on magnetic glass particles (MGP): After protease treatment and lysis of plasma samples the nucleic acid is captured by the MGP's in a generic way. The most important parameters HIV, HBV and HCV are specifically bound to the glass surface. DNA and RNA.-targets can be extracted equally well. The recovery of the nucleic acid (virus/bacteria) is 70%. After elution at elevated temperature an eluate is obtained that contains the nucleic acid in a pure solution ready for PCR reaction. The analytical sensitivity for the overall specimen preparation process (95% confidence interval) is in the range of 50-100 copies/ml plasma. The whole process is accessible to automation. In a standardized procedure the MGP-technology is insensitive to interference. Possible inhibitors to PCR are efficiently separated. Regarding amplification/ detection the homogeneous TaqMan® format allows the combination of the two steps eliminating the risk of contamination. A key issue is the simultaneous amplification and detection of several targets (HIV, HBV, HCV). An optimized multiplex TaqMan® protocol allows the detection of virus/bacteria particles down to 20-100 copies/PCR reaction depending on the parameter. The challenge is to find a compromise for the requirements of the individual targets. In summary the new developments in specimen preparation and multiplex amplification/detection for nucleic acid testing will provided further opportunities in the testing of blood/plasma for infectious parameters. Being accessible to automation they will allow cost-effective, reproducible analysis.