Strand displacement amplification (SDA)
Introduction to the BDProbeTec®ET.
A number of different amplification techniques exist [see: nucleic acid amplification tests]. Common problems include significant hands-on time, complex workflow and the potential for contamination of the work area with amplified product [amplicon]. There is a need for 'second generation', simpler, higher throughput, more user-friendly systems which incorporate real-time detection of amplified product within a sealed system, thereby minimizing opportunity for contamination of other samples by amplicon. The Becton Dickinson ProbeTecET® is a commercial attempt to achieve this; [see Little et al., 1999 below for details of the process].
In essence, the system uses isothermal strand displacement technology [Hellyer et al., 1999; Spears et al., 1997] to simultaneously amplify C. trachomatis or N. gonorrhoeae DNA present in a clinical specimen [ for the original description of the strand displacement assay the online paper of Walker et al., 1992C. trachomatis, the cryptic chlamydial plasmid; for N. gonorrhoeae, the chromosomal pilin gene-inverting protein homologue. Both these genes are present in their respective organisms in multiple copies, thereby increasing the possibility of detection. is recommended]. The target genes are: for
Performing the test
Urethral specimens are collected on rayon urethral swabs; endocervical specimens are collected on polyurethane tipped swabs. Samples are expressed into 2 mls of lysis diluent and heated to release nucleic acid. Urine samples are collected in a plastic sterile container together with special reagents provided by the manufacturer to remove amplification inhibitors. To avoid the need for specialist workers, the system is simplified, using pre-dispensed reagent devices, a programmable, expandable-spacing pipettor and an 8 x 12 sealed-microwell format and a plate reader. For each 8 x 12 tray of specimens, a positive and negative control is included for parallel processing. Thus, for a C. trachomatis test, a 96-well plate will contain one positive control (which also acts as a control for the reagents), one negative control, and up to 46 samples. Each of these 48 wells has a corresponding amplification control well directed against a different DNA sequence in order to determine if any of the specimens contain amplification inhibitors. Where specimens are being tested for both chlamydiae and gonorrhoea, one plate contains: a positive control; a negative control; and up to 30 samples. Each of the 30 samples and two controls requires three microwells: one for C. trachomatis; one for gonococci; and one for an amplification control [Little et al., 1999]. Amplification and detection takes one hour, during which a fluorochrome-labelled probe binds to the amplicons as they are produced and can be detected in real time by fluorescence energy transfer (ET) measurement using a specialised plate reader. As few as 10–15 gonococcal cells or C. trachomatis elementary bodies can be detected reliably in 1 hour, with potential throughput up to 564 patient results per typical shift [Little et al., 1999].
In practical terms the performance characteristics of the ProbeTecET® seem to be similar to the other leading nucleic acid amplification-based technologies. Little et al., 1999 tested 122 patient specimens obtained from a family practice clinic with the BD ProbeTecET® and the Abbott LCx® system for C. trachomatis and N. gonorrhoeae. The BD ProbeTecET® results for both organisms were 100% sensitive and 100% specific relative to the LCx.
Another study compared test results for gonococcal and chlamydial infection from the BD ProbeTecET® and the Roche Amplicor® PCR for 825 male and 399 female urines. It was concluded that the performance characteristics of the two tests were similar. The Becton Dickinson test was able to produce 300 results in an eight hour shift [Chan et al., 2000].
A third study compared three commercially available nucleic acid amplification tests for N. gonorrhoeae and C. trachomatis. Roche Amplicor® PCR and Becton Dickinson ProbeTecET® tests were performed on 733 endocervical swab specimens from commercial sex workers. The Abbott LCx® ligase chain reaction test was performed on a subset of 396 samples. Endocervical specimens from all women were also tested by conventional culture for N. gonorrhoeae and by Syva MicroTrak® enzyme immunoassay for C. trachomatis. All three amplification-based tests were similarly accurate and they were superior to N. gonorrhoeae or C. trachomatis antigen EIA. The Becton Dickinson ProbeTecET® SDA was the only amplification assay with 100% specificity for the detection of both N. gonorrhoeae and C. trachomatis in endocervical specimens [van Dyck et al., 2001].
A study in Glasgow, Scotland compared the performance of the LCR test with that of the Becton Dickinson ProbeTecET® SDA. The study population were 715 male and 291 female patients at a city-centre genitourinary medicine clinic and the test specimens were first catch urines from both sexes plus two endocervical swabs from the women. Discordant results were resolved using an in-house nested polymerase chain reaction. Samples positive in two out of the three assays were considered positive. The prevalence of C. trachomatis infection was high at 9.2% in men and 9.1% in women. In male urines the sensitivity of the ProbeTecET® assay was 95.5%, specificity 100%, +ve predictive value 100% and -ve predictive value 99.5%. The corresponding figures for female urines were sensitivity 77.3%, specificity 100%, +ve predictive value 100% and -ve predictive value 97.3%. For cervical swabs the results were sensitivity 90.9%, specificity100%, +ve predictive value 100% and -ve predictive value 97.3%. Thus, In a high-prevalence population, the ProbeTecET® assay proved an effective method for the detection of C. trachomatis in male urine, in cervical swabs, but not in female urine. However, further studies in lower-prevalence populations are indicated [McCartney et al., 2001].
A large multi-centre evaluation of the Becton Dickinson ProbeTecET® reported on specimens collected from 2,109 men and women attending sexually transmitted disease, family planning, or obstetrics and gynaecology clinics. Both swab and urine samples were collected, and the results obtained from 4,131 specimens were compared to those from culture and the Abbott LCx® nucleic acid amplification test. Sensitivity and specificity were calculated both with and without use of the amplification control, with little apparent difference. Without the amplification control, ProbeTecET® sensitivity for C. trachomatis and N. gonorrhoeae was 92.8% and 96.6%, respectively, for cervical swabs and 80.5% and 84.9% for female urine. For men, the comparable figures were 92.5% and 98.5%, respectively, for male urethral swabs and 93.1% and 97.9% for male urine. It was concluded that the ProbeTecET® had performance characteristics similar to the Abbot LCx® . Indeterminate results are more frequent when testing urine as opposed to cervical swabs; it is critical to ensure that, following centrifugation, the urine supernatant with its inhibitors is efficiently removed [van der Pol et al., 2001].
[ Comment: The studies above suggest that the PCR, LCR and SDA amplification-based tests have essentially similar performance except for female urine, where the ProbeTecET® sensitivity appears to be inadequate. Whether the strand displacement reaction is more susceptible to inhibitors in female urine remains to be determined; this is still a new test. However, sensitivity and specificity are not the only parameters to be considered. Other factors include cost, laboratory staff resource and training, and the number and type of specimens which can be tested at one time by the particular test format. The ProbeTecET is a relatively easy to perform and rapid assay].
MeWard May, 2002
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Chan, E. L., Brandt, K., Olienus, K., Antonishyn, N. & Horsman, G. B. (2000). Performance characteristics of the Becton Dickinson ProbeTec System for direct detection of Chlamydia trachomatis and Neisseria gonorrhoeae in male and female urine specimens in comparison with the Roche Cobas System. Archives of Pathology Laboratory Medicine 124, 1649 - 1652.
Hellyer, T. J., DesJardin, L. E., Teixeira, L., Perkins, M. D., Cave, M. D. & Eisenach, K. D. (1999). Detection of viable Mycobacterium tuberculosis by reverse transcriptase-strand displacement amplification of mRNA. Journal of Clinical Microbiology 37, 518 - 523. Full article.
Little, M. C., Andrews, J., Moore, R., Bustos, S., Jones, L., Embres, C., et al. (1999). Strand displacement amplification and homogeneous real-time detection incorporated in a second-generation DNA probe system, BDProbeTecET. Clinical Chemistry 45, 777 - 784. Full article.
McCartney, R. A., Walker, J. & Scoular, A. (2001). Detection of Chlamydia trachomatis in genitourinary medicine clinic attendees: comparison of strand displacement amplification and the ligase chain reaction. British Journal of Biomedical Science 58, 235 - 238.
Spears, P. A., Linn, C. P., Woodard, D. L. & Walker, G. T. (1997). Simultaneous strand displacement amplification and fluorescence polarization detection of Chlamydia trachomatis DNA. Analytical Biochemistry 247, 130 - 137.
van Der Pol, B., Ferrero, D. V., Buck-Barrington, L., Hook, E. 3rd., Lenderman, C., Quinn, T., Gaydos, C. A., Lovchik, J., Schachter, J., Moncada, J., Hall, G., Tuohy, M. J. & Jones, R. B. (2001). Multicenter Evaluation of the BDProbeTec ET System for Detection of Chlamydia trachomatis and Neisseria gonorrhoeae in Urine Specimens, Female Endocervical Swabs, and Male Urethral Swabs. Journal of Clinical Microbiology 39, 1008 - 1016. Full article
van Dyck, E., Ieven, M., Pattyn, S., van Damme, L. & Laga, M. (2001). Detection of Chlamydia trachomatis and Neisseria gonorrhoeae by Enzyme Immunoassay, Culture, and Three Nucleic Acid Amplification Tests. Journal of Clinical Microbiology 39, 1751 - 1756.
Walker, G. T., Little, M. C., Nadeau, J. G. & Shank, D. D. (1992). Isothermal in vitro amplification of DNA by a restriction enzyme/DNA polymerase system. Proceedings of the National Academy of Sciences of the U S A. 89, 392 - 396. Full article.
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