Sensitive and inexpensive digital DNA analysis by microfluidic enrichment of rolling circle amplified single-molecules.
Nucleic Acids Res
; 45(8): e59, 2017 05 05.
Article
en En
| MEDLINE
| ID: mdl-28077562
ABSTRACT
Single molecule quantification assays provide the ultimate sensitivity and precision for molecular analysis. However, most digital analysis techniques, i.e. droplet PCR, require sophisticated and expensive instrumentation for molecule compartmentalization, amplification and analysis. Rolling circle amplification (RCA) provides a simpler means for digital analysis. Nevertheless, the sensitivity of RCA assays has until now been limited by inefficient detection methods. We have developed a simple microfluidic strategy for enrichment of RCA products into a single field of view of a low magnification fluorescent sensor, enabling ultra-sensitive digital quantification of nucleic acids over a dynamic range from 1.2 aM to 190 fM. We prove the broad applicability of our analysis platform by demonstrating 5-plex detection of as little as â¼1 pg (â¼300 genome copies) of pathogenic DNA with simultaneous antibiotic resistance marker detection, and the analysis of rare oncogene mutations. Our method is simpler, more cost-effective and faster than other digital analysis techniques and provides the means to implement digital analysis in any laboratory equipped with a standard fluorescent microscope.
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Contexto en salud:
3_ND
Problema de salud:
3_neglected_diseases
/
3_zoonosis
Asunto principal:
ADN Circular
/
Farmacorresistencia Microbiana
/
Técnicas Biosensibles
/
Reacción en Cadena de la Polimerasa
/
Dispositivos Laboratorio en un Chip
/
Microscopía Fluorescente
Tipo de estudio:
Diagnostic_studies
Idioma:
En
Revista:
Nucleic Acids Res
Año:
2017
Tipo del documento:
Article
País de afiliación:
Suecia