Strand Displacement Amplification and Homogeneous Real-Time Detection Incorporated in a Second-Generation DNA Probe System, BDProbeTecET

Abstract

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® 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.

<p>Current and Future Perspectives on Isothermal Nucleic Acid Amplification Technologies for Diagnosing Infections</p>

“…100% for detection of C. trachomatis and N. gonorrhea nucleic acid 145 while another reported a specificity of 99.2% and sensitivity of 99.3% for N. gonorrhoea using BD ProbeTec TM SDA system on urine samples, in which results were obtained in less than 5 hrs with better performance than culture method. 152 Similarly, Van Der Pol et al- 153 reported sensitivity ranging from 80.5% to 98.5% when this assay was used to detect chlamydia and gonorrhoea infection in men and women using urine, cervical and urethral swabs.…”

“…The displaced strands then form the template for subsequent amplification which culminate to more rapid target amplification. 24,143 SDA product detection is possible by methods such as gel electrophoresis, [144][145][146] molecular beacons 145,147 and fluorescence methods. [148][149][150] SDA has been applied in the diagnosis of tuberculosis through reverse-transcriptase SDA.…”

“…An example is the BD ProbeTec TM ET system designed for detecting Chlamydia trachomatis in urine 145,151 and Neisseria gonorrhoea in urine. 152 Clinical studies on patient urine and swab samples with the developed SDA-based assay reported a sensitivity of 100% and specificity of Series of primer extension and displacement Figure 7 Strand displacement amplification.…”

Application of Molecular Diagnostic Techniques for Viral Testing

“…This method is an isothermal template amplification technique that can be used to detect trace amounts of DNA or RNA of a particular sequence (Becton Dickinson and Company, Sparks, MD). In its current format, strand displacement amplification occurs in two phases, target generation and exponential target amplification [22].…”

Helix loop-mediated isothermal amplification of nucleic acids

“…1 These disadvantages have limited its application to some special elds such as point-ofcare use in resource-limited areas. 2 In an effort to reduce the resource requirements of PCR, a variety of isothermal amplication techniques have been developed and applied widely, including self-sustained sequence replication reaction (3SR), 3 rolling circle amplication (RCA), 4 strand displacement amplication (SDA), 5 helicasedependent amplication (HDA), 6 loop-mediated isothermal amplication (LAMP) 7 and recombinase polymerase amplication (RPA), 8 Cross-priming amplication (CPA), 9 and Polymerase Spiral Reaction (PSR), 10 etc. 3SR, NASBA, SDA, HDA and RPA, require multiple enzymes and rigorous optimization, which may increase the assay cost.…”