Hockman Donna, Dong Ming, Zheng Hong, Kumar Sanjai, Huff Matthew D, Grigorenko Elena, Beanan Maureen, Duncan Robert
Diatherix Laboratories, LLC, Huntsville, AL, USA.
Laboratory of Emerging Pathogens, Division of Emerging and Transfusion Transmitted Diseases, Center for Biologics Evaluation and Research (CBER), Food and Drug Administration (FDA), Silver Spring, MD, USA.
J Microbiol Methods. 2017 Jan;132:76-82. doi: 10.1016/j.mimet.2016.11.005. Epub 2016 Nov 9.
Molecular diagnostic devices are increasingly finding utility in clinical laboratories. Demonstration of the effectiveness of these devices is dependent upon comparing results from clinical samples tested with the new device to an alternative testing method. The preparation of mock clinical specimens will be necessary for the validation of molecular diagnostic devices when a sufficient number of clinical specimens is unobtainable. Examples include rare pathogens, some of which are pathogens posing a biological weapon threat. Here we describe standardized steps for developers to follow for the culture and quantification of three organisms used to spike human whole blood to create mock specimens. The three organisms chosen for this study were the Live Vaccine Strain (LVS) of Francisella tularensis, surrogate for a potential biothreat pathogen, Escherichia coli, a representative Gram-negative bacterium and Babesia microti (Franca) Reichenow Peabody strain, representing a protozoan parasite. Mock specimens were prepared with blood from both healthy donors and donors with nonspecific symptoms including fever, malaise, and flu-like symptoms. There was no significant difference in detection results between the two groups for any pathogen. Testing of the mock samples was compared on two platforms, Target Enriched Multiplex-PCR (TEM-PCR™) and singleplex real-time PCR (RT-PCR). Results were reproducible on both platforms. The reproducibility demonstrated by obtaining the same results between two testing methods and between healthy and symptomatic mock specimens, indicates the standardized methods described for creating the mock specimens are valid and effective for evaluating diagnostic devices.
分子诊断设备在临床实验室中的应用越来越广泛。这些设备有效性的证明取决于将新设备检测临床样本的结果与另一种检测方法的结果进行比较。当无法获得足够数量的临床样本时，制备模拟临床标本对于分子诊断设备的验证是必要的。例子包括罕见病原体，其中一些是构成生物武器威胁的病原体。在这里，我们描述了开发人员在培养和定量三种用于向人全血中加样以创建模拟标本的生物体时应遵循的标准化步骤。本研究选择的三种生物体是土拉弗朗西斯菌的活疫苗株（LVS），一种潜在生物威胁病原体的替代物；大肠杆菌，一种代表性的革兰氏阴性菌；以及微小巴贝斯虫（弗兰卡）赖歇瑙-皮博迪株，代表一种原生动物寄生虫。模拟标本用来自健康供体和有非特异性症状（包括发热、不适和流感样症状）的供体的血液制备。两组之间对任何病原体的检测结果均无显著差异。在两个平台上对模拟样本进行了检测，即靶向富集多重聚合酶链反应（TEM-PCR™）和单重实时聚合酶链反应（RT-PCR）。两个平台上的结果均可重复。通过在两种检测方法之间以及健康和有症状的模拟标本之间获得相同结果所证明的可重复性，表明所描述的用于创建模拟标本的标准化方法对于评估诊断设备是有效且可靠的。
