Strand displacement amplification and homogeneous real-time detection incorporated in a second-generation DNA probe system, BDProbeTecET.

BACKGROUND Amplified DNA probes provide powerful tools for the detection of infectious diseases, cancer, and genetic diseases. Commercially available amplification systems suffer from low throughput and require decontamination schemes, significant hands-on time, and specially trained laboratory staff. Our objective was to develop a DNA probe system to overcome these limitations. METHODS We developed a DNA probe system, the BDProbeTecTMET, based on simultaneous strand displacement amplification and real-time fluorescence detection. The system uses sealed microwells to minimize the release of amplicons to the environment. To avoid the need for specially trained labor, the system uses a simple workflow with predispensed reagent devices; a programmable, expandable-spacing pipettor; and the 96-microwell format. Amplification and detection time was 1 h, with potential throughput up to 564 patient results per shift. We tested 122 total patient specimens obtained from a family practice clinic with the BD ProbeTecET and the Abbott LCx(R) amplified system for the detection of Chlamydia trachomatis and Neisseria gonorrhoeae. RESULTS Based on reportable results, the BDProbeTecET results for both organisms were 100% sensitive and 100% specific relative to the LCx. CONCLUSIONS The BDProbeTecET is an easy-to-use, high-throughput, closed amplification system for the detection of nucleic acid from C. trachomatis and N. gonorrhoeae and other organisms.

[1]  J. Hartley,et al.  Use of uracil DNA glycosylase to control carry-over contamination in polymerase chain reactions. , 1990, Gene.

[2]  C. R. Connell,et al.  Allelic discrimination by nick-translation PCR with fluorogenic probes. , 1993, Nucleic acids research.

[3]  S. Falkow,et al.  A common plasmid of Chlamydia trachomatis. , 1986, Plasmid.

[4]  R. Abramson,et al.  Detection of specific polymerase chain reaction product by utilizing the 5'----3' exonuclease activity of Thermus aquaticus DNA polymerase. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[5]  J. Fyfe,et al.  Neisseria gonorrhoeae contains multiple copies of a gene that may encode a site-specific recombinase and is associated with DNA rearrangements. , 1998, Gene.

[6]  K. Beavis,et al.  Evaluation of automated COBAS AMPLICOR PCR system for detection of several infectious agents and its impact on laboratory management , 1996, Journal of clinical microbiology.

[7]  G. Walker,et al.  Strand displacement amplification--an isothermal, in vitro DNA amplification technique. , 1992, Nucleic acids research.

[8]  D. Shank,et al.  Isothermal in vitro amplification of DNA by a restriction enzyme/DNA polymerase system. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[9]  S. Kwok,et al.  Avoiding false positives with PCR , 1989, Nature.

[10]  C. Spargo,et al.  Detection of M. tuberculosis DNA using thermophilic strand displacement amplification. , 1996, Molecular and cellular probes.

[11]  G. Walker.  Empirical aspects of strand displacement amplification. , 1993, PCR methods and applications.