Molecular Diagnostics of Infectious Diseases

In DNA-based diagnostics, the polymerase chain reaction (PCR) is the most widely used DNA amplification method. To enable both sensitive and specific detection of agents causing infectious diseases, the PCR needs to be combined with methods to prepare the clinical sample containing the genetic material of the pathogen. Furthermore, methods for detection and DNA sequence analysis of the PCR amplification products are needed. This thesis describes the development of integrated systems for detection, quantification and characterization of microorganisms. An immunomagnetic separation (IMS) technique has been used to isolate Bordetella pertussis from nasopharyngeal aspirate samples. The post-PCR detection and typing of Bordetella spp. was performed by a combination of restriction enzyme analysis of the amplified pertussis toxin (PT) promoter region and a solid-phase colorimetric detection system; detection of immobilized amplified nucleic acid (DIANA). To investigate whether this approach could be used for reliable discrimination between the three Bordetella spp. infecting humans, the PT promoter region used for diagnostics was sequenced in 33 strains. To determine the DNA sequence of this polymorphic and repetitive region, a new technique, bidirectional pyrosequencing, was utilized. This procedure was used to resolve the sequence of this DNA region, which is able to form stable secondary structures in conventional Sanger DNA sequencing. A quantitative assay using competitive PCR and the DIANA detection technique was also developed, for quantification of B. pertussis in clinical samples. By arbitrary PCR, a DNA sequence apparently specific for Vibrio cholerae O139 Bengal was isolated and characterized. A nested PCR assay was developed for sensitive and specific detection of V. cholerae O139 Bengal in clinical samples and in environmental water samples, where differentiation between V. cholerae O139 Bengal and V. cholera O1 is of epidemiological interest. The magnetic separation approach was also used to capture human immunodeficiency virus (HIV-1) RNA from patient plasma. A nested reverse transcription (RT)-PCR with four internal competitors was combined with electrophoretic separation and quantification of the PCR amplicons on an automated DNA sequencer. From the internal calibration curve, the amount of HIV-1 RNA in the sample could be determined. Furthermore, a primer extension assay was combined with detection and quantification of the competitive PCR products by the same biochemiluminescent detection technique that is used in pyrosequencing. Quantification of HIV-1 viral load has implications in monitoring of antiretroviral therapy and in assessment of disease progression into AIDS.