期刊
SENSORS AND ACTUATORS B-CHEMICAL
卷 339, 期 -, 页码 -出版社
ELSEVIER SCIENCE SA
DOI: 10.1016/j.snb.2021.129873
关键词
Duplex PCR; Microfluidics; Fluorescence detection; Chlamydia trachomatis
资金
- Enterprise Ireland [CF/2014/4306A]
A PCR assay was developed to screen Chlamydia trachomatis directly from urine with high sensitivity and specificity, suitable for implementation on a disposable cartridge system. The assay protocol involved heating urine samples to 85°C for ten minutes and performing a duplex realtime PCR on resulting urine lysate.
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 ?L 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 LightCyclerTM 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.
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