Background We are developing a highly sensitive, ultra-rapid, multiplex PCR method with fully-integrated DNA preparation and ambient-stable reagents. The assay was used in conjunction with a novel electrochemical detection method to demonstrate low copy number amplification and detection in <20 min, as a point-of-care (POC) diagnostic test for Chlamydia trachomatis (CT).
Methods The method employs custom PCR cards, utilising a thin-film laminate construction to achieve rapid heat transfer, in conjunction with an ultra-rapid thermocycler. All reagents necessary to perform the extraction, amplification and detection are deposited into the cards and air dried at the point of manufacture. Novel, ambient-stable reagent formulations with an 18 month shelf life have been developed. A sample is added to the card and DNA extracted from the sample. The resulting eluate reconstitutes dried PCR reagents and a 40-cycle multiplex PCR is performed using rapid thermocycling. Amplified target is detected using electrochemically-labelled target-specific probes and a double-stranded DNA-specific exonuclease to release the electrochemical label. Released label is read by applying a voltage to a screen printed carbon electrode and at a known oxidation potential the label is oxidised producing a measurable current. The unique rapid performance of this device has been demonstrated in terms of analytical sensitivity and reagent stability under ambient storage conditions. Multiplex capability is demonstrated in this test with the presence of internal control (IC) DNA.
Results Analytical sensitivity of the device was evaluated by testing dilutions of CT in the presence of IC DNA. The results show CT detection down to 50 copies when co-extracted, amplified in duplex and detected electrochemically with the IC DNA (see graph). Tests on the reagents dried into the device showed stability for 18 months when stored at ambient temperature (20–25°C). Reagent performance after 18 months' storage was shown to be equivalent to performance at time zero see Abstract P4-S1.03 figure 1.
Conclusions The results show that this device could be used to perform ultra-rapid multiplex PCR with no user intervention after sample addition, allowing minimally-trained staff to carry out the assay in <20 min, meeting the needs for a “true” POC device. Ambient stability of the reagents negates the requirement for any specialised storage conditions.
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