Droplet Cas12a Assay Enables DNA Quantification from Unamplified Samples at the Single-Molecule Level.
Nano Lett
; 21(11): 4643-4653, 2021 06 09.
Article
em En
| MEDLINE
| ID: mdl-34038136
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.
Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Infecções por Vírus Epstein-Barr
/
Vírus da Febre Suína Africana
Limite:
Animals
Idioma:
En
Revista:
Nano Lett
Ano de publicação:
2021
Tipo de documento:
Article
País de afiliação:
China