Pathobiology and Population Sciences, Royal Veterinary College, Hawkshead, Herts, AL9 7TA, UK.
School of Biosciences, University of Nottingham, Sutton Bonington Campus, Loughborough, Leics, LE12 5RD, UK.
Microb Biotechnol. 2020 May;13(3):738-746. doi: 10.1111/1751-7915.13518. Epub 2019 Dec 3.
Here, we describe the development of a method that exploits bacteriophage D29 as a lysis agent for efficient DNA extraction from low numbers of mycobacterial cells. This method (Actiphage ) used in combination with PCR achieved rapid and sensitive (LOD ≤ 10 cell ml ) detection and identification of viable, pathogenic mycobacteria in blood samples within 6 h. We demonstrate that mycobacteriophage D29 can be used to detect a range of mycobacteria from clinical blood samples including both Mycobacterium tuberculosis complex and Mycobacterium avium subsp. paratuberculosis without the need for culture and confirms our earlier observations that a low-level bacteraemia is associated with these infections in cattle. In a study of M. bovis-infected cattle (n = 41), the sensitivity of the Actiphage method was 95 % (95 % CI; 0.84-0.99) and specificity was 100 % (95% CI; 0.92-1). We further used Actiphage to demonstrate viable Mycobacterium avium subsp. paratuberculosis is present in the blood of Johne's infected cattle. This method provides a revolutionary new tool for the study of infections caused by these difficult to grow pathogens.
在这里，我们描述了一种方法的开发，该方法利用噬菌体 D29 作为裂解剂，从少量分枝杆菌细胞中有效提取 DNA。该方法（Actiphage）与 PCR 联合使用，可在 6 小时内快速、灵敏（LOD≤10 细胞/ml）检测和鉴定血液样本中的活性病原体分枝杆菌。我们证明，分枝杆菌噬菌体 D29 可用于检测临床血液样本中的一系列分枝杆菌，包括结核分枝杆菌复合体和禽分枝杆菌副结核亚种，而无需培养，并证实了我们之前的观察结果，即这些感染与牛的低水平菌血症有关。在一项感染牛分枝杆菌的牛研究中（n=41），Actiphage 方法的敏感性为 95%（95%CI；0.84-0.99），特异性为 100%（95%CI；0.92-1）。我们进一步使用 Actiphage 来证明在感染 Johne 病的牛的血液中存在活的禽分枝杆菌副结核亚种。该方法为研究这些难以培养的病原体引起的感染提供了一种全新的革命性工具。
