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Sensitive fluorescent detection of Listeria monocytogenes by combining a universal asymmetric polymerase chain reaction with rolling circle amplification.
- Source :
-
Journal of Pharmaceutical & Biomedical Analysis . May2019, Vol. 169, p181-187. 7p. - Publication Year :
- 2019
-
Abstract
- Graphical abstract Highlights • A fluorescence assay has been developed for L. monocytogenes detection by combining aPCR and RCA. • By using universal sequence in aPCR reaction, the assay could be applied for other bacteria detection. • The assay showed an LOD of 4.8 × 101 CFU/mL in pure culture and 4.0 × 102 CFU/g in lettuce. Abstract A new, facile, low-cost, and highly sensitive method for detection of Listeria monocytogenes involving a combination of asymmetric polymerase chain reaction (aPCR) and rolling circle amplification (RCA) had been developed. The aPCR-RCA processes were not new but components of the processes made the assay useful. Twenty-one thymine (21-T) tagged forward primer generated universal twenty-one adenine (21-A) aPCR amplicons after aPCR amplification. A poly-T sequence dumbbell-like RCA template was produced through the blunt-end ligation activity of T4 DNA ligase. After the mixture of aPCR amplicons and dumbbell-like RCA template, the RCA reaction would initiate when the addition of phi29 DNA polymerase, then a large number of G-quadruplex sequences were produced which allowed the intercalation of Thioflavin T (3,6-dimethyl-2-(4-dimethylaminophenyl) benzo-thiazolium cation, THT) for easy fluorescence detection. Under the optimal conditions, the assay showed a limit of detection (LOD) of 4.8 × 101 CFU/mL in pure culture and 4.0 × 102 CFU/g in spiked lettuce homogenates. By changing the aPCR primer, the aPCR-RCA method developed in this study had a potential to detect other bacteria without the design an RCA template for each bacterium. [ABSTRACT FROM AUTHOR]
Details
- Language :
- English
- ISSN :
- 07317085
- Volume :
- 169
- Database :
- Academic Search Index
- Journal :
- Journal of Pharmaceutical & Biomedical Analysis
- Publication Type :
- Academic Journal
- Accession number :
- 135686146
- Full Text :
- https://doi.org/10.1016/j.jpba.2019.03.016