tetano
Editor, Senior Moderator
Anal Chem
. 2021 Nov 25.
doi: 10.1021/acs.analchem.1c04065. Online ahead of print.
Universally Stable and Precise CRISPR-LAMP Detection Platform for Precise Multiple Respiratory Tract Virus Diagnosis Including Mutant SARS-CoV-2 Spike N501Y
Tong Zhang[SUP] 1 [/SUP], Wei Zhao[SUP] 1 [/SUP], Wang Zhao[SUP] 1 [/SUP], Yuying Si[SUP] 2 [/SUP], Nianzhen Chen[SUP] 2 [/SUP], Xi Chen[SUP] 1 [/SUP], Xinlian Zhang[SUP] 1 [/SUP], Lieying Fan[SUP] 2 [/SUP], Guodong Sui[SUP] 1 3 4 [/SUP]
Affiliations
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.
. 2021 Nov 25.
doi: 10.1021/acs.analchem.1c04065. Online ahead of print.
Universally Stable and Precise CRISPR-LAMP Detection Platform for Precise Multiple Respiratory Tract Virus Diagnosis Including Mutant SARS-CoV-2 Spike N501Y
Tong Zhang[SUP] 1 [/SUP], Wei Zhao[SUP] 1 [/SUP], Wang Zhao[SUP] 1 [/SUP], Yuying Si[SUP] 2 [/SUP], Nianzhen Chen[SUP] 2 [/SUP], Xi Chen[SUP] 1 [/SUP], Xinlian Zhang[SUP] 1 [/SUP], Lieying Fan[SUP] 2 [/SUP], Guodong Sui[SUP] 1 3 4 [/SUP]
Affiliations
- PMID: 34818890
- DOI: 10.1021/acs.analchem.1c04065
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.