Droplet Cas12a Assay Enables DNA Quantification from Unamplified Samples at the Single-Molecule Level.
Nano Lett
; 21(11): 4643-4653, 2021 06 09.
Article
in English
| MEDLINE | ID: covidwho-1303734
ABSTRACT
DNA quantification is important for biomedical research, but the routinely used techniques rely on nucleic acid amplification which have inherent issues like cross-contamination risk and quantification bias. Here, we report a CRISPR-Cas12a-based molecular diagnostic technique for amplification-free and absolute quantification of DNA at the single-molecule level. To achieve this, we first screened out the optimal reaction parameters for high-efficient Cas12a assay, yielding over 50-fold improvement in sensitivity compared with the reported Cas12a assays. We further leveraged the microdroplet-enabled confinement effect to perform an ultralocalized droplet Cas12a assay, obtaining excellent specificity and single-molecule sensitivity. Moreover, we demonstrated its versatility and quantification capability by direct counting of diverse virus's DNAs (African swine fever virus, Epstein-Barr virus, and Hepatitis B virus) from clinical serum samples with a wide range of viral titers. Given the flexible programmability of crRNA, we envision this amplification-free technique as a versatile and quantitative platform for molecular diagnosis.
Keywords
Full text:
Available
Collection:
International databases
Database:
MEDLINE
Main subject:
Epstein-Barr Virus Infections
/
African Swine Fever Virus
Type of study:
Diagnostic study
/
Prognostic study
/
Randomized controlled trials
Limits:
Animals
Language:
English
Journal:
Nano Lett
Year:
2021
Document Type:
Article
Affiliation country:
Acs.nanolett.1c00715
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