In DNA-based diagnostics, the polymerase chain reaction(PCR) is the most widely used DNA amplification method. Toenable both sensitive and specific detection of agents causinginfectious diseases, the PCR needs to becombined with methodsto prepare the clinical sample containing the genetic materialof the pathogen. Furthermore, methods for detection and DNAsequence analysis of the PCR amplification products are needed.This thesis describes the development of integrated systems fordetection, quantification and characterization ofmicroorganisms.
An immunomagnetic separation (IMS) technique has been usedto isolateBordetella pertussisfrom nasopharyngeal aspiratesamples. The post-PCR detection and typing ofBordetellaspp. was performed by a combination ofrestriction enzyme analysis of the amplified pertussis toxin(PT) promoter region and a solid-phase colorimetric detectionsystem; detection of immobilized amplified nucleic acid(DIANA). To investigate whether this approach could be used forreliable discrimination between the threeBordetellaspp. infecting humans, the PT promoter regionused for diagnostics was sequenced in 33 strains. To determinethe DNA sequence of this polymorphic and repetitive region, anew technique, bidirectional pyrosequencing, was utilized. Thisprocedure was used to resolve the sequence of this DNA region,which is able to form stable secondary structures inconventional Sanger DNA sequencing. A quantitative assay usingcompetitive PCR and the DIANA detection technique was alsodeveloped, for quantification ofB. pertussisin clinical samples.
By arbitrary PCR, a DNA sequence apparently specific forVibrio choleraeO139 Bengal was isolated andcharacterized. A nested PCR assay was developed for sensitiveand specific detection ofV. choleraeO139 Bengal in clinical samples and inenvironmental water samples, where differentiation betweenV. choleraeO139 Bengal andV. choleraO1 is of epidemiological interest.
The magnetic separation approach was also used to capturehuman immunodeficiency virus (HIV-1) RNA from patient plasma. Anested reverse transcription (RT)-PCR with four internalcompetitors was combined with electrophoretic separation andquantification of the PCR amplicons on an automated DNAsequencer. From the internal calibration curve, the amount ofHIV-1 RNA in the sample could be determined. Furthermore, aprimer extension assay was combined with detection andquantification of the competitive PCR products by the samebiochemiluminescent detection technique that is used inpyrosequencing. Quantification of HIV-1 viral load hasimplications in monitoring of antiretroviral therapy and inassessment of disease progression into AIDS.
Key words:bioluminescence,Bordetella, competitive PCR, DNA sequencing, humanimmunodeficiency virus type 1, PCR, pyrosequencing, solid-phasetechnology,Vibrio cholerae
© Malin Nygren, 2000
Stockholm: Bioteknologi , 2000. , 69 p.