C-terminal part of glutamate-ammonia-ligase adenyltransferase gene identified by RAPD-HRM with 3H primer for E. coli screening

Title:

C-terminal part of glutamate-ammonia-ligase adenyltransferase gene identified by RAPD-HRM with 3H primer for E. coli screening

Creator:

Chen, Y. C., Lai, Y. S., Shyu, D. J. H., Chang, Y. W. , Chen, Z. R., Liao, Y. K., Pang, C. T., and Chang, K. T.

Identifier:

https://cdk.lib.cas.cz/client/handle/uuid:7cb7ce9b-1ad9-46f1-b87b-bbaed24a3f10
uuid:7cb7ce9b-1ad9-46f1-b87b-bbaed24a3f10

Subject:

E. coli, random-amplified-polymorphic-DNA, 3H primer, high-resolution melt, and sequence characterized amplified (region)

Type:

model:article and TEXT

Format:

bez média and svazek

Description:

A single random oligonucleotide 3H primer has been previously applied in random-amplfied-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, O83:H1, O104:H4, O157:H7 and O169:H41. 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. and Corresponding author: Ko-Tung Chang

Language:

English

Rights:

http://creativecommons.org/publicdomain/mark/1.0/
policy:public

Coverage:

[88]-100

Source:

Folia biologica | 2019 Volume:65 | Number:2

Harvested from:

CDK

Metadata only:

false