C-Terminal Part of Glutamate-Ammonia-Ligase Adenyltransferase Gene Identified by RAPD-HRM with 3H Primer for <i>E. Coli</i> Screening

Chen, Y. C.; Lai, Y. S.; Shyu, D. J. H.; Chang, Y. W.; Chen, Z. R.; Liao, Y. K.; Pang, C. T.; Chang, Ko-Tung

doi:10.14712/fb2019065020088

Folia Biologica > Archive > 2019, Vol. 65 > Issue 2 > C-Terminal Part of…

Fol. Biol. 2019, 65, 88-100

https://doi.org/10.14712/fb2019065020088

C-Terminal Part of Glutamate-Ammonia-Ligase Adenyltransferase Gene Identified by RAPD-HRM with 3H Primer for E. Coli Screening

Y. C. Chen¹, Y. S. Lai^1,2, D. J. H. Shyu¹, Y. W. Chang¹, Z. R. Chen¹, Y. K. Liao¹, C. T. Pang¹, Ko-Tung Chang^1,2,3

¹Department of Biological Science and Technology, National Pingtung University of Science and Technology, Pingtung, Taiwan
²Research Center for Animal Biologics, National Pingtung University of Science and Technology, Pingtung, Taiwan
³Flow Cytometry Center, Precision Instruments Center, Office of Research and Development, National Pingtung University of Science and Technology, Pingtung, Taiwan

Received March 2019
Accepted April 2019

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, 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.

Keywords

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

Funding

This study was supported by the Ministry of Science and Technology in Taiwan (grants MOST 106-2314-B-020-001, MOST 104-2314-B-020-001-MY3 and MOST 108-3017-F-020-001).