Application of CRISPR-Cas12a Enhanced Fluorescence Assay Coupled with Nucleic Acid Amplification for the Sensitive Detection of African Swine Fever Virus.
ACS Synth Biol
; 9(9): 2339-2350, 2020 09 18.
Article
in En
| MEDLINE
| ID: mdl-32786346
ABSTRACT
African swine fever (ASF) is one of the most severe diseases of pigs. In this study, a CRISPR-Cas12a (also known as Cpf1) system coupled with nucleic acid amplification was optimized for the detection of ASF virus (ASFV). Two novel single-stranded DNA-fluorophore-quencher (ssDNA-FQ) reporters were developed to increase the brightness of the fluorescent signal for the visualization of nucleic acid detection. The CRISPR-Cas12a system was used to simultaneously cleave the polymerase chain reaction (PCR) or loop-mediated isothermal amplification (LAMP) amplicons and the newly developed ssDNA-FQ reporter, resulting in fluorescence that could be easily detected in multiple platforms, especially on cheap and portable blue or UV light transilluminators. This specific cleavage with fluorescence reveals the presence of the amplicon and confirms its identity, thereby preventing false-positive test results from nonspecific amplicons. This method is also uninterfered by the presence of large amounts of irrelevant background DNA and displays no cross-reactivity with other porcine DNA or RNA viruses. When coupled with LAMP, the Cas12a platform can detect a plasmid containing p72 with as few as 2 copies/µL reaction. Our results indicate that the CRISPR-Cas12a enhanced fluorescence assay coupled with nucleic acid amplification is robust, convenient, specific, confirmatory, affordable, and potentially adaptable for ASF diagnosis.
Key words
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Swine
/
DNA, Viral
/
Nucleic Acid Amplification Techniques
/
African Swine Fever Virus
/
CRISPR-Cas Systems
Type of study:
Diagnostic_studies
Limits:
Animals
Language:
En
Journal:
ACS Synth Biol
Year:
2020
Document type:
Article