Development of a point-of-care rapid multiplex assay for simultaneous detection of common respiratory pathogens in clinical samples using CRISPR-based readouts

Project Abstract

We present the development and validation of a point-of-care (POC) rapid multiplex assay capable of simultaneous detection of common respiratory pathogens directly from clinical samples, utilizing CRISPR-based readouts to achieve high specificity, sensitivity, and rapid turnaround times suitable for frontline settings. The assay integrates isothermal nucleic acid amplification with programmable CRISPR-Cavage or CRISPR-Cas12/13 detection modalities to enable simultaneous identification of target pathogens, including influenza A/B, respiratory syncytial virus (RSV), SARS-CoV-2, human metapneumovirus (hMPV), adenoviruses, parainfluenza viruses, and rhinoviruses. A streamlined sample processing workflow minimizes handling steps, employing a minimal-resource extraction-free protocol compatible with saliva and nasopharyngeal swab specimens. The detection system leverages fluorescence- or lateral-flow-based readouts, driven by collateral cleavage activity of Cas enzymes upon target recognition, with signal amplification achieved through multiplexed guide RNAs and carefully optimized reporter molecules to reduce cross-reactivity and false positives. The study optimized primer and guide RNA design to discriminate highly conserved regions while avoiding sequence homology cross-reactivity among related pathogens. Analytical performance was evaluated using quantified reference materials to establish limits of detection (LOD) for each pathogen, with an overall multiplex LOD in the low copies per reaction range. Specificity testing against a panel of non-target respiratory organisms demonstrated high selectivity. The assay was further validated with a diverse clinical sample set (n > 400), comprising well-characterized positive and negative specimens, to assess diagnostic sensitivity, specificity, positive predictive value, and negative predictive value against gold-standard RT-qPCR assays. Results indicate concordance with existing laboratory methods and robust performance across varying viral loads, sample matrices, and storage conditions. Operationally, the POC device was engineered for portability, low power consumption, and ease of use, featuring a single-step reaction setup, isothermal amplification at approximately 37–42°C, and an integrated detection module compatible with smartphone-based readout or compact fluorescence readers. Data were analyzed using an open-source software framework enabling real-time result interpretation, multiplex pattern recognition, and automated reporting to clinical information systems. The workflow supports rapid decision-making in outpatient clinics, emergency departments, and field settings, enabling timely initiation of isolation protocols and antiviral therapies. Limitations identified include potential interference from high mucosal viscosity samples, the need for ongoing surveillance to account for pathogen evolution, and the requirement for periodic revalidation to maintain assay accuracy. Ethical considerations addressed informed consent and data privacy, with de-identified results used for performance assessment. Overall, the study demonstrates the feasibility and clinical utility of a CRISPR-based multiplex POC assay for comprehensive respiratory pathogen detection, offering a scalable framework for rapid, accurate, and decentralized diagnostics in respiratory illness outbreaks.

Project Overview

What This Project Is About

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A straightforward study of a quick, home-friendly test that can check for several common respiratory germs at once, using a CRISPR-based method to signal if any are present in a sample like a throat swab or spit.

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The Problem It Addresses

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Currently, doctors often test for one pathogen at a time, which takes longer and may delay treatment. This project aims to combine several tests into one rapid tool, saving time and resources while helping patients get faster, more accurate care.

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Objectives of the Project

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1. Explain the concept of a point-of-care test and why multiplexing matters.
2. Describe how CRISPR-based signaling can indicate the presence of multiple pathogens.
3. Prototype a simple assay that can detect several respiratory pathogens in a single run.
4. Assess the test’s speed, accuracy, and ease of use under lab-like conditions.
5. Identify practical limits, such as false positives/negatives and sample handling.

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What You Will Do Step by Step

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1. Review background material on respiratory pathogens and CRISPR readouts.

2. Design a basic multiplex test plan (which targets to include and how signals are read).

3. Build a simple assay using available lab kits to simulate detection signals.

4. Test with known samples to check accuracy and speed.

5. Analyze results to identify strengths and limits, and suggest improvements.

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Expected Outcome

The project should deliver a clear description of a multiplex CRISPR-based point-of-care approach, practical performance data, and recommendations for further development toward real-world use. It aims to show feasibility, identify challenges, and provide a path for a more robust, field-ready test.