Development of cpn60-Based Real-Time Quantitative PCR Assays for the Detection of 14 Campylobacter Species and Application to Screening of Canine Fecal Samples
Development of cpn60-Based Real-Time Quantitative PCR Assays for the Detection of 14 Campylobacter Species and Application to Screening of Canine Fecal Samples
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DOI：
10.1128/aem.00101-09
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发表时间：
2009-05-15
影响因子：
4.4
通讯作者：
Hill, Janet E.
中科院分区：
文献类型：
--
作者：
Chaban, Bonnie;Musil, Kristyna M.;Hill, Janet E.
弯曲杆菌属物种是人类和动物健康中的重要生物。弯曲菌的鉴定目前需要从复杂的样品中生长出生物体并进行生化鉴定。在许多情况下，待测样品的条件和/或许多弯曲杆菌属物种的挑剔性质限制了实验室环境中弯曲杆菌的检测。为了解决这个问题，我们设计了一套实时定量PCR（qPCR）检测和定量14种弯曲杆菌，C。coli、C. concisus、C. curvus、弯角隐翅虫C. fetus、C. gracilis、C. helveticus，C. hyoelasticum，C. jejuni、C. lari，C. mucosalis、C. rectus、C. showae，C. Acutorum和C.乌普萨拉人，直接从粪便中提取的DNA。通过使用cpn 60（也称为hsp 60或groEL）基因的一个区域，该基因编码普遍保守的60-kDa伴侣蛋白，设计并验证了物种特异性测定。然后采用这些试验确定犬粪便样本中弯曲杆菌属的流行率。粪便样本中含有可检测和可定量的C。fetus、C. gracilis、C. helveticus，C. jejuni、C. showae和C. upsaliensis，大多数样品含有多种弯曲杆菌属物种。本研究是C. fetus、C. gracilis、C. mucosalis和C. Showae检测在狗和牵连狗作为这些物种的水库。所描述的qPCR测定为研究人员提供了一种新的工具，以不依赖于培养的方式研究许多弯曲杆菌属物种。
Campylobacter species are important organisms in both human and animal health. The identification of Campylobacter currently requires the growth of organisms from complex samples and biochemical identification. In many cases, the condition of the sample being tested and/or the fastidious nature of many Campylobacter species has limited the detection of campylobacters in a laboratory setting. To address this, we have designed a set of real-time quantitative PCR (qPCR) assays to detect and quantify 14 Campylobacter species, C. coli, C. concisus, C. curvus, C. fetus, C. gracilis, C. helveticus, C. hyointestinalis, C. jejuni, C. lari, C. mucosalis, C. rectus, C. showae, C. sputorum, and C. upsaliensis, directly from DNA extracted from feces. By use of a region of the cpn60 (also known as hsp60 or groEL) gene, which encodes the universally conserved 60-kDa chaperonin, species-specific assays were designed and validated. These assays were then employed to determine the prevalence of Campylobacter species in fecal samples from dogs. Fecal samples were found to contain detectable and quantifiable levels of C. fetus, C. gracilis, C. helveticus, C. jejuni, C. showae, and C. upsaliensis, with the majority of samples containing multiple Campylobacter species. This study represents the first report of C. fetus, C. gracilis, C. mucosalis, and C. showae detection in dogs and implicates dogs as a reservoir for these species. The qPCR assays described offer investigators a new tool to study many Campylobacter species in a culture-independent manner.