Rational Programming of Cas12a for Early-Stage Detection of COVID-19 by Lateral Flow Assay and Portable Real-Time Fluorescence Readout Facilities

Coronavirus disease 2019 (COVID-19) caused by the SARS-CoV-2 virus has led to a global pandemic with a high spread rate and pathogenicity. Thus, with limited testing solutions, it is imperative to develop early-stage diagnostics for rapid and accurate detection of SARS-CoV-2 to contain the rapid transmission of the ongoing COVID-19 pandemic. In this regard, there remains little knowledge about the integration of the CRISPR collateral cleavage mechanism in the lateral flow assay and fluorophotometer. In the current study, we demonstrate a CRISPR/Cas12a-based collateral cleavage method for COVID-19 diagnosis using the Cas12a/crRNA complex for target recognition, reverse transcription loop-mediated isothermal amplification (RT-LAMP) for sensitivity enhancement, and a novel DNA capture probe-based lateral flow strip (LFS) or real-time fluorescence detector as the parallel system readout facility, termed CRICOLAP. Our novel approach uses a customized reporter that hybridizes an optimized complementary capture probe fixed at the test line for naked-eye result readout. The CRICOLAP system achieved ultra-sensitivity of 1 copy/µL in ~32 min by portable real-time fluorescence detection and ~60 min by LFS. Furthermore, CRICOLAP validation using 60 clinical nasopharyngeal samples previously verified with a commercial RT-PCR kit showed 97.5% and 100% sensitivity for S and N genes, respectively, and 100% specificity for both genes of SARS-CoV-2. CRICOLAP advances the CRISPR/Cas12a collateral cleavage result readout in the lateral flow assay and fluorophotometer, and it can be an alternative method for the decentralized field-deployable diagnosis of COVID-19 in remote and limited-resource locations.

Rapid Detection of blaKPC, blaNDM, blaOXA-48-like and blaIMP Carbapenemases in Enterobacterales Using Recombinase Polymerase Amplification Combined With Lateral Flow Strip

The emergence of carbapenemase-producing Enterobacterales (CPE) infections is a major global public health threat. Rapid and accurate detection of pathogenic bacteria is essential to optimize treatment and timely avoid further transmission of these bacteria. Here, we aimed to develop a rapid on site visualization detection method for CPE using improved recombinase polymerase amplification (RPA) combined with lateral flow strip (LFS) method, based on four most popular carbapenemase genes: blaKPC, blaNDM, blaOXA-48-like, and blaIMP. All available allelic variants of the above carbapenemases were downloaded from the β-lactamase database, and the conserved regions were used as targets for RPA assay. Five primer sets were designed targeting to each carbapenemase gene and the RPA amplification products were analyzed by agarose gel electrophoresis. FITC-labeled specific probes were selected, combined with the best performance primer set (Biotin-labeled on the reverse primer), and detected by RPA-LFS. Mismatches were made to exclude the false positive signals interference. This assay was evaluated in 207 clinically validated carbapenem-resistant Enterobacterales (CRE) isolates and made a comparison with conventional PCR. Results showed that the established RPA-LFS assay for CPE could be realized within 30 min at a constant temperature of 37°C and visually detected amplification products without the need for special equipment. This assay could specifically differentiate the four classes of carbapenemases without cross-reactivity and shared a minimum detection limit of 100 fg/reaction (for blaKPC, blaNDM, and blaOXA-48-like) or 1000 fg/reaction (for blaIMP), which is ten times more sensitive than PCR. Furthermore, the detection of 207 pre-validated clinically CRE strains using the RPA-LFS method resulted in 134 blaKPC, 69 blaNDM, 3 blaOXA-48-like, and 1 blaIMP. The results of the RPA-LFS assay were in consistent with PCR, indicating that this method shared high sensitivity and specificity. Therefore, the RPA-LFS method for CPE may be a simple, specific, and sensitive method for the rapid diagnosis of carbapenemase Enterobacterales.

Development and Performance verification of colloidal gold labeled SARS-CoV-2 antigen detection method for routine popular screening of COVID-19 with clinical samples in Poland and China

In this research, we have constructed and optimized the colloidal gold labeled lateral flow strip (LFS) for rapid detection of antigen of SARS-CoV-2 and rapid screening of COVID-19. Based on the constructed and optimized colloidal gold lateral flow strip, the parameters of the LFS have been well evaluated with the clinical samples in the professional labs. The screening performance have also been evaluated from the aspects including the CT values, age distribution and onset of symptoms. Finally, based on the detection results of 420 clinical samples, the LFS can achieve the screening of COVID-19 with the positive percentage agreement (PPA, sensitivity), negative percent agreement (NPA, specificity), the positive predictive value (PPV) and the negative predictive value (NPV) of 96.8%, 100%, 100% and 96.6%, respectively, indicating the powerful potential for practical screening applications in pandemic control. Of great significance, this developed SARS-CoV-2 antigen detection method has also been successfully utilized for screening of delta-variant of SARS-CoV-2.

Assessing the Use of DNA Detection Platforms Combined With Passive Wind-Powered Spore Traps for Early Surveillance of Potato and Tomato Late Blight in Canada

Quantitative PCR (qPCR), loop-mediated amplification (LAMP), and lateral flow strip-based recombinase polymerase amplification (RPA-LFS) assays were assessed for early detection of Phytophthora infestans, the global causal agent of potato and tomato late blight, on passive wind-powered spore traps known as Spornados. Spore traps were deployed in potato and tomato fields during the 2018, 2019, and 2020 growing seasons in the provinces of Alberta, British Columbia, Manitoba, Prince Edward Island, and Ontario. All assays used DNA extracts from Spornado cassette membranes targeting the P. infestans nuclear ribosomal internal transcribed spacer. A total of 1,003 Spornado samples were qPCR tested, yielding 115 positive samples for P. infestans spores. In further assessment of these samples, LAMP detected P. infestans in 108 (93.9%) of 115 qPCR positive samples, and RPA-LFS detected it in 103 (89.6%). None of the assays showed cross-reaction with other Phytophthora species or pathogenic fungi known to infect potato and tomato. The qPCR detected ≤1 fg of P. infestans DNA, and LAMP and RPA-LFS amplified 10 fg in as little as 10 min. All assays detected P. infestans before the first report of late blight symptoms in commercial potato or tomato fields within each region or province. The combination of Spornado passive samplers with qPCR, LAMP, or RPA-LFS proved a valuable spore trapping system for early surveillance of late blight in potato and tomato. Both LAMP and RPA-LFS showed potential as alternative approaches to qPCR for in-field monitoring of P. infestans.