Abstract
A rapid and accurate on-site diagnostic test for pathogens including influenza viruses is critical for preventing the spread of infectious diseases. Two types of influenza virus, A and B cause seasonal flu epidemics, whereas type A can cause influenza pandemics. To specifically detect influenza A (IAV) and B (IBV) viruses, we developed a clustered, regularly interspaced, short palindromic repeats (CRISPR) and CRISPR-associated (Cas) system-based assay. By coupling reverse transcription recombinase polymerase amplification (RT-RPA) and reverse transcription loop-mediated isothermal amplification (RT-LAMP), a CRISPR-Cas12a DNA endonuclease-targeted CRISPR trans-reporter (DETECTR) detected IAV and IBV titers as low as 1 × 100 plaque forming units (PFUs) per reaction without exhibiting cross-reactivity. Only 75 to 85 min were required to detect IAV and IBV, depending on isothermal nucleic acid amplification methods, and results were verified using a lateral flow strip assay that does not require additional analytic equipment. Taken together, our findings establish RT-RPA and RT-LAMP-coupled DETECTR-based diagnostic tests for rapid, specific and high-sensitivity detection of IAV and IBV using fluorescence and lateral flow assays. The diagnostic test developed in this study can be used to distinguish IAV and IBV infections, a capability that is necessary for monitoring and preventing the spread of influenza epidemics and pandemics.
Highlights
Rapid and accurate diagnosis is critical for preventing the spread of infectious diseases.Influenza viruses are a significant public health concern and a major cause of epidemics and pandemics, including the 1918 and 2009 pandemics
We developed a method for detecting influenza A virus (IAV) and influenza B virus (IBV) using the DNA endonuclease-targeted CRISPR trans-reporter (DETECTR) assay together with reverse transcription recombinase polymerase amplification (RT-recombinase polymerase amplification (RPA)) and reverse transcription loop-mediated isothermal amplification (RT-loop-mediated isothermal amplification (LAMP)) and applied a lateral flow assay for rapid interpretation of results without the need for any analytical equipment
After RT-RPA and RT-LAMP, amplicons were detected by DETECTR assay followed by fluorescence or lateral flow assay
Summary
Influenza viruses are a significant public health concern and a major cause of epidemics and pandemics, including the 1918 and 2009 pandemics. There are three types of influenza viruses that infect humans, which are distinguished based on their matrix (M) or nucleoprotein (NP) genes: influenza A virus (IAV), influenza B virus (IBV) and influenza C virus (ICV); of these IAV and IBV cause seasonal flu epidemics. IAV can be transmitted between animals and humans and is extremely variable through reassortment of viral genes between subtypes from different species, contributing to its ability to cause influenza pandemics [1]. A rapid diagnostic test to distinguish between IAV and IBV infections is necessary for preventing the spread of influenza viruses
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