Ciara Kathleen O'SUllivan

The increasing spread of toxin-producing Gambierdiscus dinoflagellates has led to cases of ciguatera poisoning in previously nonendemic regions. Ciguatoxins (CTXs) are potent, heat-stable compounds responsible for ciguatera, and their rapid and reliable analysis in seafood is essential to prevent illness. This study presents the development of the first lateral flow assay (LFA) for the detection of major Pacific CTXs congeners. The assay employs previously developed monoclonal antibodies in a sandwich format: specific antibodies against CTX1B and CTX3C are immobilized on a single test line, while the reporter antibody is a single antibody recognizing major Pacific CTX congeners that is conjugated to gold nanoparticles. The LFA provides one-step visual results in 30 min, making it suitable for resource-limited settings. It exhibits a visual limit of detection of 400 pg/mL and the assay is highly specific, showing no cross-reactivity with other marine toxins. It successfully detected Pacific CTXs spiked in noncontaminated fish extracts, as well as in naturally contaminated samples as previously confirmed by LC-MS/MS, with an estimated cutoff of approximately 0.1 μg/kg. Overall, this LFA demonstrates great potential as a rapid screening tool for outbreak investigations and research programs.

Quantum biosensors, which harness quantum effects to detect biomarkers, could address the urgent need for more sensitive rapid diagnostics. Lateral flow tests using nitrogen-vacancy centres in nanodiamond labels offer high sensitivity and robustness by controlling the spin-dependent fluorescence to remove background. This is particularly important in complex and variable clinical samples. However, to date only model systems have been studied with few clinical samples. Here we show results of a clinical evaluation of a spin-enhanced nanodiamond test for SARS-CoV-2 antigen with 103 upper respiratory tract swab samples. We find 95.1% sensitivity (Ct ≤ 30) and 100% specificity benchmarked against RT-qPCR, with no cross-reactivity to influenza A, RSV, and Rhinovirus. Modelling with patient data yields a mean of 2.0-days earlier detection compared to conventional gold-nanoparticle tests (just 0.6 days after RT-qPCR) with 2.2-fold more patients detected on the first day of symptom onset, potentially reducing the transmission risk and protecting populations.

A new enzymatic method for truncation of aptamers from 5’‐end has been developed and demonstrated on a newly selected indole‐modified β ‐conglutin aptamer. This method relies on extension of a primer containing ribonucleotides, which can be specifically hydrolyzed by RNase, thereby removing the whole or 5’‐part of the primer. This approach enables flexible synthesis of modified aptamers truncated from the 5’ end, 3’ end, or both ends—a capability previously achievable only through chemical oligonucleotide synthesis. Furthermore, by employing doubly labeled primers, diverse labeled or modified nucleotides are introduced to prepare 5’‐labeled aptamers suitable for fluorescence‐ or immobilization‐based assays. The different variations of this method also enable the synthesis of mutated sequences to study the effect of modifications and their positions. Overall, this enzymatic method provides a valuable alternative for the truncation or labeling of (hyper)modified and functional oligonucleotides avoiding the need for chemical synthesis.

In line with the global research priorities on sexually transmitted infections (STIs), we developed a molecular point-of-care test (POCT) for the parasite Trichomonas vaginalis that causes the STI trichomoniasis. Trichomoniasis remains the most common curable nonviral STI. We report on the use of specific T. vaginalis single-stranded-tailed DNA primers in combination with recombinase polymerase amplification (RPA) in a protein-free nucleic acid lateral flow (NALF) device for use at the point of care. The use of aminated DNA probes eliminates the need for proteins (such as streptavidin or hapten-specific antibodies) for detection of the DNA amplicons, simplifying the manufacturing process and improving reproducibility and cost-effectiveness. The estimated shelf life of the NALF devices is at least 6.6 months at 22 °C, and the devices exhibited high reproducibility. The RPA-NALF requires three simple operator steps with minimal instrumentation and takes approximately 30 min from sample preparation to interpretation of the result. It is specific to T. vaginalis and can detect 1.3 × 10³ cells/mL with visual readout or 282 cells/mL with the aid of a portable LFA reader. Analysis of the two sets of clinical genomic DNA extracts showed that RPA-NALF is positive for all samples positive by culture assay and for samples with Cq ≤ 23 with the S-DiaMGTV qPCR. Finally, RPA-NALF is positive on biobanked vaginal swabs with T. vaginalis Cq ≤ 25 with the Allplex qPCR assays. These results demonstrate that RPA-NALF can specifically detect a moderate load of T. vaginalis even in the presence of other microbial DNA and cells and other components of clinical samples.

Competitive LFAs are widely employed to detect targets ranging from small molecules to large proteins across diverse fields. This review explores their function using theoretical models and critically addresses factors affecting their performance.

Aptamer lateral flow assay (ALFA) for testing T. vaginalis (Tv) in cervicovaginal lavage (CVL).