C-Terminal Part of Glutamate-Ammonia-Ligase Adenyltransferase Gene Identified by RAPD-HRM with 3H Primer for E. Coli Screening.
Folia Biol (Praha)
; 65(2): 88-100, 2019.
Article
em En
| MEDLINE
| ID: mdl-31464184
ABSTRACT
A single random oligonucleotide 3H primer has been previously applied in random-amplified- polymorphic-DNA (RAPD)-PCR to distinguish stocked bacteria E. coli within a cocktail mixture also containing Enterococcus faecalis, Bifidobacterium longum and Ruminococcus gnavus. In this study, we demonstrate that a 702 base pair (bp) gene fragment can be amplified as a unique pattern by RAPD-PCR using a 3H primer in human faeces containing E. coli. This unique 702 bp amplicon contained a 687 bp gene fragment identified as the C-terminal region of the glutamate-ammonia-ligase adenyltransferase (glnE) gene of E. coli. By high-resolution melt (HRM) analysis, a mean melt-curve temperature of this 702 bp amplicon was determined to be approximately 88.1 ± 0.22 degrees Celsius (°C). A combination of RAPD with HRM in one single reaction based on this amplicon can achieve semi-quantitative detection of up to 102 CFU/ml of E. coli. To increase the signal intensity of HRM, a primer pair capable of screening E. coli directly from fresh human faeces was re-designed from the 687 bp gene segment, giving a mean peak melt-curve temperature at 88.35 ± 0.11 °C. Finally, single-nucleotide polymorphisms of this 687 bp gene segment were analysed for pathogenic E. coli strains, including UMN026, O83H1, O104H4, O157H7 and O169H41. We conclude that this 687 bp segment of the glnE gene has a high potential for screening of human faecal E. coli, including pathogenic strains, in contaminated food and water.
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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Primers do DNA
/
Técnica de Amplificação ao Acaso de DNA Polimórfico
/
Escherichia coli
/
Genes Bacterianos
/
Glutamato-Amônia Ligase
Tipo de estudo:
Diagnostic_studies
/
Screening_studies
Limite:
Humans
Idioma:
En
Revista:
Folia Biol (Praha)
Ano de publicação:
2019
Tipo de documento:
Article
País de afiliação:
Taiwan