Development of a disposable cartridge real-time PCR test for detection of Chlamydia trachomatis directly from urine
| Autor: | Desmond Brennan, Terry J. Smith, Martin Cormican, Eoin Clancy, Louise O'Connor, James Kileen, Paul Galvin |
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| Rok vydání: | 2021 |
| Předmět: |
Chromatography
Chemistry Pcr assay Metals and Alloys 02 engineering and technology Urine 010402 general chemistry 021001 nanoscience & nanotechnology Condensed Matter Physics medicine.disease_cause 01 natural sciences 0104 chemical sciences Surfaces Coatings and Films Electronic Optical and Magnetic Materials law.invention Cartridge Real-time polymerase chain reaction law Materials Chemistry medicine Electrical and Electronic Engineering 0210 nano-technology Chlamydia trachomatis Instrumentation Polymerase chain reaction |
| Zdroj: | Sensors and Actuators B: Chemical. 339:129873 |
| ISSN: | 0925-4005 |
| Popis: | We present a PCR assay screening Chlamydia trachomatis directly from urine without pre-amplification or sample clean-up and demonstrate suitability for implementation on a disposable cartridge system. Primers and probes were designed to detect all Chlamydia trachomatis serovars with a high degree of sensitivity and specificity. The assay also incorporated a synthetic internal amplification control to monitor inhibition of the PCR reaction. The assay protocol worked directly on urine samples with initial heating to 85 °C for ten minutes, followed by a duplex realtime PCR (95 °C, 55 °C, 45 cycles) on resulting urine lysate. The test cartridge incorporated heating and liquid manipulation using pinch valves and a compressible blister pouch. Lyophilised PCR reagents were incorporated into the cartridge and were re-suspended by a 40 μL aliquot of the urine lysate. Primers were designed to screen for all Chlamydia trachomatis serovars with a high degree of sensitivity and specificity. Evaluation of the newly developed PCR assay was carried out on the LightCycler™ using urine samples previously confirmed positive or negative for the target pathogen, using the ABBOTT real-time PCR Cobas CT/NG test at a local hospital. |
| Databáze: | OpenAIRE |
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