Acharya Kamal R, Dhand Navneet K, Whittington Richard J, Plain Karren M
Sydney School of Veterinary Science, Faculty of Science, University of SydneyCamden, NSW, Australia; Department of Livestock Services, Regional Veterinary Diagnostic LaboratoryDhangadhi, Nepal.
Sydney School of Veterinary Science, Faculty of Science, University of Sydney Camden, NSW, Australia.
Front Microbiol. 2017 Feb 2;8:115. doi: 10.3389/fmicb.2017.00115. eCollection 2017.
Molecular tests such as polymerase chain reaction (PCR) are increasingly being applied for the diagnosis of Johne's disease, a chronic intestinal infection of ruminants caused by subspecies (MAP). Feces, as the primary test sample, presents challenges in terms of effective DNA isolation, with potential for PCR inhibition and ultimately for reduced analytical and diagnostic sensitivity. However, limited evidence is available regarding the magnitude and diagnostic implications of PCR inhibition for the detection of MAP in feces. This study aimed to investigate the presence and diagnostic implications of PCR inhibition in a quantitative PCR assay for MAP (High-throughput Johne's test) to investigate the characteristics of samples prone to inhibition and to identify measures that can be taken to overcome this. In a study of fecal samples derived from a high prevalence, endemically infected cattle herd, 19.94% of fecal DNA extracts showed some evidence of inhibition. Relief of inhibition by a five-fold dilution of the DNA extract led to an average increase in quantification of DNA by 3.3-fold that consequently increased test sensitivity of the qPCR from 55 to 80% compared to fecal culture. DNA extracts with higher DNA and protein content had 19.33 and 10.94 times higher odds of showing inhibition, respectively. The results suggest that the current test protocol is sensitive for herd level diagnosis of Johne's disease but that test sensitivity and individual level diagnosis could be enhanced by relief of PCR inhibition, achieved by five-fold dilution of the DNA extract. Furthermore, qualitative and quantitative parameters derived from absorbance measures of DNA extracts could be useful for prediction of inhibitory fecal samples.
诸如聚合酶链反应（PCR）之类的分子检测方法越来越多地用于诊断副结核分枝杆菌（MAP）引起的反刍动物慢性肠道感染——副结核病。粪便作为主要检测样本，在有效分离DNA方面存在挑战，存在PCR抑制的可能性，并最终导致分析和诊断灵敏度降低。然而，关于PCR抑制对粪便中MAP检测的影响程度和诊断意义的证据有限。本研究旨在调查在用于MAP的定量PCR检测（高通量副结核病检测）中PCR抑制的存在情况及其诊断意义，以研究易于产生抑制作用的样本特征，并确定可采取的克服此问题的措施。在一项对来自高流行率、地方性感染牛群的粪便样本的研究中，19.94%的粪便DNA提取物显示出某种抑制迹象。将DNA提取物五倍稀释后抑制作用得到缓解，导致DNA定量平均增加3.3倍，因此与粪便培养相比，qPCR的检测灵敏度从55%提高到80%。DNA和蛋白质含量较高的DNA提取物出现抑制的几率分别高出19.33倍和10.94倍。结果表明，当前的检测方案对副结核病的牛群水平诊断是敏感的，但通过将DNA提取物五倍稀释来缓解PCR抑制作用，可以提高检测灵敏度和个体水平诊断。此外，从DNA提取物吸光度测量得出的定性和定量参数可能有助于预测具有抑制作用的粪便样本。
