Smartphone-Based SARS-CoV-2 and Variants Detection System using Colorimetric DNAzyme Reaction Triggered by Loop-Mediated Isothermal Amplification (LAMP) with Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)

Abstract

Coronavirus disease (COVID-19) has affected people for over two years. Moreover, the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants has raised concerns regarding its accurate diagnosis. Here, we report a colorimetric DNAzyme reaction triggered by loop-mediated isothermal amplification (LAMP) with clustered regularly interspaced short palindromic repeats (CRISPR), referred to as DAMPR assay for detecting SARS-CoV-2 and variants genes with attomolar sensitivity within an hour. The CRISPR-associated protein 9 (Cas9) system eliminated false-positive signals of LAMP products, improving the accuracy of DAMPR assay. Further, we fabricated a portable DAMPR assay system using a three-dimensional printing technique and developed a machine learning (ML)-based smartphone application to routinely check diagnostic results of SARS-CoV-2 and variants. Among blind tests of 136 clinical samples, the proposed system successfully diagnosed COVID-19 patients with a clinical sensitivity and specificity of 100% each. More importantly, the D614G (variant-common), T478K (delta-specific), and A67V (omicron-specific) mutations of the SARS-CoV-2 S gene were detected selectively, enabling the diagnosis of 70 SARS-CoV-2 delta or omicron variant patients. The DAMPR assay system is expected to be employed for on-site, rapid, accurate detection of SARS-CoV-2 and its variants gene and employed in the diagnosis of various infectious diseases.

Emerging Multiplex Nucleic Acid Diagnostic Tests for Combating COVID-19

“…For example, upon binding to ssDNA or ssRNA, the trans -cleavage mechanism can release a fluorescence reporter from the quenched fluorescence probe [ 9 , 12 , 20 , 70 , 71 , 72 , 73 , 74 , 75 , 76 ]. Different CRISPR-Cas systems have been utilized for multiplex SARS-CoV-2 detection, including the Cas9 system (recognizes dsDNA) [ 77 , 78 ], Cas12 (recognizes both ssDNA and dsDNA) [ 9 , 71 , 72 , 75 , 76 , 79 ], and Cas13 (recognizes ssRNA) [ 12 , 20 , 71 , 74 , 80 , 81 ]. Additionally, amplification techniques, such as RT-PCR [ 9 , 71 , 72 , 76 ], RT-LAMP [ 73 , 77 , 79 ], and RT-RPA [ 9 , 20 , 75 , 78 ], are often combined with CRISPR-based detection to enhance the limit of detection (LOD) in viral detection assays.…”

“…To double check specific mutation genes, CRISPR/Cas9 with crRNA specifically designed to recognize the target genes (WT or MT) will cleave the G-quadruplex DNAzyme, which turns off the colorimetric signal. For multiplexing, the on/off colorimetric signals of multiple genes tested in parallel were derived to determine the VOCs [ 77 ].…”

“…CRISPR-based multiplexing assays may be conducted in a one-pot reaction targeting ~2–8 target genes at once [ 75 , 78 ]. However, to improve multiplexing capabilities, many groups utilized CRISPR/Cas reactions running separately in parallel with crRNAs specifically designed to target mutation genes using different platform designs [ 9 , 70 , 71 , 77 ]. For example, Welch et al developed a combined RT-PCR CRISPP-based diagnostic and microfluidic panel, mCARMEN.…”

Principles and Applications of Loop-Mediated Isothermal Amplification to Point-of-Care Tests

“…As smartphones are emerging as the most promising monitoring devices, providing portability, affordability, and rapidity, they can be used as readout [ 107 , 108 , 109 , 110 ], image-processing [ 111 , 112 ], or operating tools [ 55 , 113 ] on diagnostic platforms. Furthermore, LAMP has been applied in diagnostic systems using smartphones owing to its various monitoring mechanisms, including color change, fluorescence, and turbidity, which can be implemented on smartphones.…”

“…Recently, Song et al reported a smartphone application based on machine learning for checking diagnostic results [ 111 ]. The diagnosis of SARS-CoV-2 and its variants consists of a colorimetric DNAzyme reaction triggered by a LAMP amplicon with clustered regularly interspaced short palindromic repeats (CRISPR).…”