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. 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. 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.

中文翻译：

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实时定量PCR检测方法的开发和应用，用于评估农用地表水和各种粪便中粪便和拟南芥的流行情况。

不久，粪便中的拟南芥和兰科拟南芥被确定为新兴的人类和动物病原体。在本文中，我们证明了使用直接来自rpoB和gyrA基因的A. faecis和A. lanthieri的物种特异性寡核苷酸引物对开发和优化两种基于DNA的直接定量实时PCR分析方法。最初，通过熔解曲线分析来验证并确认引物的特异性和每个靶标参照菌株的扩增子大小。使用高压灭菌的农业地表水和奶牛粪便样品中已知数量的加标农杆菌和兰参参考菌株获得的最小定量限为100个细胞mL-1或g-1，建立标准曲线。验证了每种特定物种的qPCR检测方法，并将其应用于确定流行率，并量化了农业主导和非农业参考流域的天然地表水中每种目标物种的细胞总数。此外，确定了人类和各种动物（例如，狗，猫，奶牛和家禽）粪便样本的患病率和密度。总体而言，粪便中地表水和粪便的流行率分别为21％和28％。粪便中水和粪便的最大浓度分别为100毫升-1和2.3×107细胞g-1。在较低的频率（2％）中检测到兰色曲霉，其地表水中的最大浓度为4.2×105细胞100 mL-1；粪便样本的患病率和最大密度分别为10％和2.0×106个细胞g-1。结果表明，这些物种在农业地表水中的发生可能是由于粪便样品中检测到了这两种物种而导致的粪便污染了牲畜，人类或野生生物中的水。新的实时qPCR分析可以在3小时内快速准确地进行检测，以量化各种复杂环境样品中存在的粪曲霉和兰氏曲霉细胞的总数。