Real-time quantitative PCR assay development and application for assessment of agricultural surface water and various fecal matter for prevalence of Aliarcobacter faecis and Aliarcobacter lanthieri.
Agriculture
Animals
Bacterial Proteins
Campylobacteraceae
/ classification
Cattle
DNA Gyrase
/ genetics
DNA Primers
/ genetics
DNA-Directed RNA Polymerases
/ genetics
Humans
Livestock
/ microbiology
Manure
/ microbiology
Prevalence
Real-Time Polymerase Chain Reaction
/ methods
Species Specificity
Water Microbiology
Agricultural watershed
Aliarcobacter faecis
Aliarcobacter lanthieri
Assay
Fecal matter
Surface water
qPCR
Journal
BMC microbiology
ISSN: 1471-2180
Titre abrégé: BMC Microbiol
Pays: England
ID NLM: 100966981
Informations de publication
Date de publication:
16 06 2020
16 06 2020
Historique:
received:
03
03
2020
accepted:
18
05
2020
entrez:
18
6
2020
pubmed:
18
6
2020
medline:
27
5
2021
Statut:
epublish
Résumé
Aliarcobacter faecis and Aliarcobacter lanthieri are recently identified as emerging human and animal pathogens. In this paper, we demonstrate the development and optimization of two direct DNA-based quantitative real-time PCR assays using species-specific oligonucleotide primer pairs derived from rpoB and gyrA genes for A. faecis and A. lanthieri, respectively. Initially, the specificity of primers and amplicon size of each target reference strain was verified and confirmed by melt curve analysis. Standard curves were developed with a minimum quantification limit of 100 cells mL Each species-specific qPCR assay was validated and applied to determine the rate of prevalence and quantify the total number of cells of each target species in natural surface waters of an agriculturally-dominant and non-agricultural reference watershed. In addition, the prevalence and densities were determined for human and various animal (e.g., dogs, cats, dairy cow, and poultry) fecal samples. Overall, the prevalence of A. faecis for surface water and feces was 21 and 28%, respectively. The maximum A. faecis concentration for water and feces was 2.3 × 10 The results indicate that the occurrence of these species in agricultural surface water is potentially due to fecal contamination of water from livestock, human, or wildlife as both species were detected in fecal samples. The new real-time qPCR assays can facilitate rapid and accurate detection in < 3 h to quantify total numbers of A. faecis and A. lanthieri cells present in various complex environmental samples.
Sections du résumé
BACKGROUND
Aliarcobacter faecis and Aliarcobacter lanthieri are recently identified as emerging human and animal pathogens. In this paper, we demonstrate the development and optimization of two direct DNA-based quantitative real-time PCR assays using species-specific oligonucleotide primer pairs derived from rpoB and gyrA genes for A. faecis and A. lanthieri, respectively. Initially, the specificity of primers and amplicon size of each target reference strain was verified and confirmed by melt curve analysis. Standard curves were developed with a minimum quantification limit of 100 cells mL
RESULTS
Each species-specific qPCR assay was validated and applied to determine the rate of prevalence and quantify the total number of cells of each target species in natural surface waters of an agriculturally-dominant and non-agricultural reference watershed. In addition, the prevalence and densities were determined for human and various animal (e.g., dogs, cats, dairy cow, and poultry) fecal samples. Overall, the prevalence of A. faecis for surface water and feces was 21 and 28%, respectively. The maximum A. faecis concentration for water and feces was 2.3 × 10
CONCLUSIONS
The results indicate that the occurrence of these species in agricultural surface water is potentially due to fecal contamination of water from livestock, human, or wildlife as both species were detected in fecal samples. The new real-time qPCR assays can facilitate rapid and accurate detection in < 3 h to quantify total numbers of A. faecis and A. lanthieri cells present in various complex environmental samples.
Identifiants
pubmed: 32546238
doi: 10.1186/s12866-020-01826-3
pii: 10.1186/s12866-020-01826-3
pmc: PMC7298852
doi:
Substances chimiques
Bacterial Proteins
0
DNA Primers
0
Manure
0
DNA-Directed RNA Polymerases
EC 2.7.7.6
DNA Gyrase
EC 5.99.1.3
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
164Subventions
Organisme : Agriculture and Agri-food Canada
ID : J-000157 and J-001012
Pays : International
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