Using Cy5-dUTP labelling of RPA-amplicons with downstream microarray analysis for the detection of antibiotic resistance genes.
Sci Rep
; 11(1): 20137, 2021 10 11.
Article
en En
| MEDLINE
| ID: mdl-34635776
ABSTRACT
In this report we describe Cy5-dUTP labelling of recombinase-polymerase-amplification (RPA) products directly during the amplification process for the first time. Nucleic acid amplification techniques, especially polymerase-chain-reaction as well as various isothermal amplification methods such as RPA, becomes a promising tool in the detection of pathogens and target specific genes. Actually, RPA even provides more advantages. This isothermal method got popular in point of care diagnostics because of its speed and sensitivity but requires pre-labelled primer or probes for a following detection of the amplicons. To overcome this disadvantages, we performed an labelling of RPA-amplicons with Cy5-dUTP without the need of pre-labelled primers. The amplification results of various multiple antibiotic resistance genes indicating great potential as a flexible and promising tool with high specific and sensitive detection capabilities of the target genes. After the determination of an appropriate rate of 1% Cy5-dUTP and 99% unlabelled dTTP we were able to detect the blaCTX-M15 gene in less than 1.6E-03 ng genomic DNA corresponding to approximately 200 cfu of Escherichia coli cells in only 40 min amplification time.
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 Bacteriano
/
Farmacorresistencia Microbiana
/
Carbocianinas
/
Proteínas de Escherichia coli
/
Nucleótidos de Desoxiuracil
/
Escherichia coli
/
Antibacterianos
Tipo de estudio:
Diagnostic_studies
Idioma:
En
Revista:
Sci Rep
Año:
2021
Tipo del documento:
Article
País de afiliación:
Alemania