检测双芽巴贝斯虫的实时 PCR 检测方法的建立与比较：三个实验室间的一致性研究（培养法和常规 PCR）。
Development and comparison of a real-time PCR assay for detection of Dichelobacter nodosus with culturing and conventional PCR: harmonisation between three laboratories.
机构信息
Department of Bacteriology, National Veterinary Institute, SE-751 89 Uppsala, Sweden.
出版信息
Acta Vet Scand. 2012 Jan 31;54(1):6. doi: 10.1186/1751-0147-54-6.
BACKGROUND
Ovine footrot is a contagious disease with worldwide occurrence in sheep. The main causative agent is the fastidious bacterium Dichelobacter nodosus. In Scandinavia, footrot was first diagnosed in Sweden in 2004 and later also in Norway and Denmark. Clinical examination of sheep feet is fundamental to diagnosis of footrot, but D. nodosus should also be detected to confirm the diagnosis. PCR-based detection using conventional PCR has been used at our institutes, but the method was laborious and there was a need for a faster, easier-to-interpret method. The aim of this study was to develop a TaqMan-based real-time PCR assay for detection of D. nodosus and to compare its performance with culturing and conventional PCR.
METHODS
A D. nodosus-specific TaqMan based real-time PCR assay targeting the 16S rRNA gene was designed. The inclusivity and exclusivity (specificity) of the assay was tested using 55 bacterial and two fungal strains. To evaluate the sensitivity and harmonisation of results between different laboratories, aliquots of a single DNA preparation were analysed at three Scandinavian laboratories. The developed real-time PCR assay was compared to culturing by analysing 126 samples, and to a conventional PCR method by analysing 224 samples. A selection of PCR-products was cloned and sequenced in order to verify that they had been identified correctly.
RESULTS
The developed assay had a detection limit of 3.9 fg of D. nodosus genomic DNA. This result was obtained at all three laboratories and corresponds to approximately three copies of the D. nodosus genome per reaction. The assay showed 100% inclusivity and 100% exclusivity for the strains tested. The real-time PCR assay found 54.8% more positive samples than by culturing and 8% more than conventional PCR.
CONCLUSIONS
The developed real-time PCR assay has good specificity and sensitivity for detection of D. nodosus, and the results are easy to interpret. The method is less time-consuming than either culturing or conventional PCR.
背景
绵羊腐蹄病是一种具有世界范围发生的传染性疾病。主要病原体是挑剔的 Dichelobacter nodosus 细菌。在斯堪的纳维亚，腐蹄病于 2004 年在瑞典首次被诊断出来，后来在挪威和丹麦也有发现。对羊蹄进行临床检查是诊断腐蹄病的基础，但也应检测到 D. nodosus 以确认诊断。我们研究所使用了基于聚合酶链反应（PCR）的检测方法，包括常规 PCR，但该方法繁琐，需要一种更快、更容易解释的方法。本研究旨在开发一种针对 D. nodosus 的 TaqMan 实时 PCR 检测方法，并比较其与培养和常规 PCR 的性能。
方法
设计了一种针对 16S rRNA 基因的 D. nodosus 特异性 TaqMan 实时 PCR 检测方法。使用 55 株细菌和 2 株真菌菌株测试了该方法的包容性和排他性（特异性）。为了评估不同实验室之间结果的敏感性和协调性，在三个斯堪的纳维亚实验室分析了单个 DNA 制剂的等分试样。通过分析 126 个样本将开发的实时 PCR 检测方法与培养法进行了比较，并通过分析 224 个样本与常规 PCR 方法进行了比较。选择了一些 PCR 产物进行克隆和测序，以验证它们已被正确识别。
结果
开发的检测方法的检测限为 3.9 fg D. nodosus 基因组 DNA。这一结果在所有三个实验室都得到了验证，相当于每个反应中约有三个 D. nodosus 基因组拷贝。该检测方法对测试菌株具有 100%的包容性和 100%的排他性。实时 PCR 检测方法比培养法发现了 54.8%更多的阳性样本，比常规 PCR 方法发现了 8%更多的阳性样本。
结论
开发的实时 PCR 检测方法对 D. nodosus 的检测具有良好的特异性和敏感性，结果易于解释。该方法比培养法或常规 PCR 法耗时更少。
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