Lateral flow nitrocellulose strip-based rapid tests (LFTs) have become a popular and cost-effective way to detect infections. However, these tests have limited sensitivity, further reducing their relevance in diagnostics.1 A strategy to overcome these limitations is to develop fluorescence-based tests, using fluorescent particles such as semiconductor quantum dots (QDs). QDs are extremely bright and can be conjugated with proteins and nucleic acids, making them an attractive alternative to organic fluorophores.2 Recent advancements in QDs chemistry have introduced particles that are more stable in buffers,3 finally making it possible to develop sensitive QD-based LFTs.
We have used QDs to develop prototype LFTs for the detection of human and animal Chlamydia from clinical samples with minimal sample processing. We used isothermal amplification (performed on a heating block) of the plasmid and CpecG_0573 genes for C. trachomatis and C. pecorum4, respectively, using biotin and digoxigenin labelled primers. End-point result detection was performed using a benchtop fluorescent reader. These rapid tests operated as sandwich assays, where bioconjugated QDs formed fluorescent lines in presence of the targeted amplicon.
The performance of the QD-based LFTs was compared to reference tests such as isothermal amplification performed in a real-time fluorometer and qPCR, respectively. The end-point isothermal assay had analytical sensitivities of 50 genome copies, comparable to that of currently used isothermal and qPCR assays, but faster (30 min total assay time, compared to 90 – 120 min for qPCR). The target specificity was confirmed against a range of DNA extracts from Gram positive and negative bacteria, human gDNA, and related chlamydial species.
Finally, we also evaluated the use of a portable, custom-made heating device and a hand-held fluorescent reading aid that can be used with QD-based LFT to unambiguously determine chlamydial infections in field settings. Our results demonstrate the potential of QD-based LFTs as a more sensitive and accurate alternative to traditional LFDs for medical diagnostics, perfectly suitable for field setting.