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
Autor: | Izhar U. H. Khan, Emilia Craiovan, Mary G. Miltenburg, Graham Wilkes, David R. Lapen, Edward Topp, Michel Cloutier |
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Rok vydání: | 2020 |
Předmět: |
Microbiology (medical)
Veterinary medicine Livestock lcsh:QR1-502 Assay Biology Real-Time Polymerase Chain Reaction Microbiology Melting curve analysis lcsh:Microbiology 03 medical and health sciences Aliarcobacter lanthieri Bacterial Proteins Species Specificity Prevalence Animals Humans Campylobacteraceae Feces 030304 developmental biology DNA Primers 0303 health sciences 030306 microbiology Aliarcobacter faecis Agriculture Surface water DNA-Directed RNA Polymerases rpoB Amplicon Size Fecal coliform Standard curve Manure qPCR Fecal matter DNA Gyrase Agricultural watershed Cattle Water Microbiology Cow dung Research Article |
Zdroj: | BMC Microbiology BMC Microbiology, Vol 20, Iss 1, Pp 1-13 (2020) |
ISSN: | 1471-2180 |
Popis: | 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− 1 or g− 1 obtained using known quantities of spiked A. faecis and A. lanthieri reference strains in autoclaved agricultural surface water and dairy cow manure samples. 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 × 107 cells 100 mL- 1 and 1.2 × 107 cells g− 1, respectively. A. lanthieri was detected at a lower frequency (2%) with a maximum concentration in surface water of 4.2 × 105 cells 100 mL− 1; fecal samples had a prevalence and maximum density of 10% and 2.0 × 106 cells g− 1, respectively. 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 A. faecis and A. lanthieri cells present in various complex environmental samples. |
Databáze: | OpenAIRE |
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