Development and evaluation of MMIRA-CRISPR/Cas13a-MBQD assay for the detection of influenza A/B viruses and SARS-CoV-2

AimSensitive and convenient methodologies for detection of influenza A/B viruses and SARS-CoV-2 are of great importance for further disease control and treatment. This study aimed to establish a rapid, sensitive and specialized instrument-free method for the detection of these viruses.Materials & methodsWe developed a multiplex Multienzyme Isothermal Rapid Amplification (MIRA) assay for simultaneous amplification of influenza A/B viruses and SARS-CoV-2. The amplicons were further detected by CRISPR/Cas13a. And the signal was finally detected using a novel magnetic bead-quantum dot (MBQD) sensor based on the integrity of the Link-Probe.ResultsThe established integrated assay (known as MMIRA-CRISPR/Cas13a-MBQD) could be completed within 60 minutes, and the limit of detection (LOD) was determined to be 1, 2, and 56 copies of RNA for SARS-CoV-2, influenza A, and influenza B, respectively. No cross-reactivity with other respiratory pathogens was found. Clinical sample analysis showed the MMIRA-CRISPR/Cas13a-MBQD assay had an overall sensitivity and specificity of 98.8% and 100%, respectively. Good agreement was observed between the assay and real-time RT-PCR (p = 0.988).ConclusionThe established assay is a promising tool for the detection of influenza A/B viruses and SARS-CoV-2. No expensive instrument required makes this assay more suitable for use in resource-limited settings.