Universally Stable and Precise CRISPR-LAMP Detection Platform for Precise Multiple Respiratory Tract Virus Diagnosis Including Mutant SARS-CoV-2 Spike N501Y

Abstract

Nowadays, rapid and accurate diagnosis of respiratory tract viruses is an urgent need to prevent another epidemic outbreak. To overcome this problem, we have developed a clustered, regularly interspaced short palindromic repeats (CRISPR) loop mediated amplification (LAMP) technology to detect influenza A virus, influenza B virus, respiratory syncytial A virus, respiratory syncytial B virus, and severe acute respiratory syndrome coronavirus 2, including variants of concern (B.1.1.7), which utilized CRISPR-associated protein 12a (Cas12a) to advance LAMP technology with the sensitivity increased 10 times. To reduce aerosol contamination in CRISPR-LAMP technology, an uracil-DNA-glycosylase-reverse transcription-LAMP system was also developed which can effectively remove dUTP-incorporated LAMP amplicons. In vitro Cas12a cleavage reaction with 28 crRNAs showed that there were no position constraints for Cas12a/CRISPR RNA (crRNA) recognition and cleavage in LAMP amplicons, and even the looped position of LAMP amplicons could be effectively recognized and cleaved. Wild-type or spike N501Y can be detected with a limit of detection of 10 copies/μL (wild-type) even at a 1% ratio level on the background (spike N501Y). Combining UDG-RT-LAMP technology, CRISPR-LAMP design, and mutation detection design, we developed a CRISPR-LAMP detection platform that can precisely diagnose pathogens with better stability and significantly improved point mutation detection efficiency.

Paper Device Combining CRISPR/Cas12a and Reverse-Transcription Loop-Mediated Isothermal Amplification for SARS-CoV-2 Detection in Wastewater

“…Notably, in terms of on-site qualitative testing, false positives caused by nonspecific amplification interfere with the test results . As shown in Figure a–c, stripes appeared at the front end in lines 2 and 3, which were formed by small DNA fragments, indicating that there were dimers in the negative control and samples with concentrations below the LODs.…”

Highly Specific and Rapid Detection of Hepatitis C Virus Using RT-LAMP-Coupled CRISPR–Cas12 Assay

“…It possesses a distinct feature known as the collateral ( trans -cleavage) activity, a nonspecific single-stranded DNA (ssDNA) cleavage activity following the recognition and cleavage of the specific target [ 14 , 15 ]. Recently, CRISPR–Cas12 combined with the RT-LAMP technique has been extensively used for the detection of RNA viruses in particular [ 16 , 17 ]. The CRISPR–Cas12 system has not yet been proposed for the detection of HCV RNA.…”

CRISPR/Cas9-based coronal nanostructures for targeted mitochondria single molecule imaging

“…CRISPR/Cas system's precise cutting of nucleic acids makes it an important molecular scissors in the process of identifying and manipulating nucleic acids 24,25 . And, in some reports, the CRISPR/Cas system can respond in a short period of time due to excellent precision and clever controllable strategies [26][27][28] . These properties make it become the candidate for fluorescent signal switches [29][30][31] .…”

“…24,25 And, in some reports, the CRISPR/Cas system can respond in a short period of time due to excellent precision and clever controllable strategies. [26][27][28] These properties make it become a candidate for uorescent signal switches. [29][30][31] But remarkably, when proteins and nucleic acids are the major components of cellular uptake, there are twosided effects.…”