U.S. Food and Drug Administration, Center for Veterinary Medicine, Office of Research, 8401 Muirkirk Road, Laurel, Maryland 20708, USA.
J Food Prot. 2012 Jun;75(6):1107-12. doi: 10.4315/0362-028X.JFP-11-415.
The U.S. Food and Drug Administration (FDA) has previously validated a real-time PCR-based assay that is currently being used by the FDA and several state laboratories as the official screening method. Due to several shortcomings to the assay, a multiplex real-time PCR assay (MRTA) to detect three ruminant species (bovine, caprine, and ovine) was developed using a lyophilized bead design. The assay contained two primer or probe sets: a "ruminant" set to detect bovine-, caprine-, and ovine-derived materials and a second set to serve as an internal PCR control, formatted using a lyophilized bead design. Performance of the assay was evaluated against stringent acceptance criteria developed by the FDA's Center for Veterinary Medicine's Office of Research. The MRTA for the detection of ruminant DNA passed the stringent acceptance criteria for specificity, sensitivity, and selectivity. The assay met sensitivity and reproducibility requirements by detecting 30 of 30 complete feed samples fortified with meals at 0.1 % (wt/wt) rendered material from each of the three ruminant species. The MRTA demonstrated 100 % selectivity (0.0 % false positives) for negative controls throughout the assessment period. The assay showed ruggedness in both sample selection and reagent preparation. Second and third analyst trials confirmed the quality of the written standard operating procedure with consistency of results. An external laboratory participating in a peer-verification trial demonstrated 100 % specificity in identifying bovine meat and bone meal, while exhibiting a 0.03 % rate of false positives. The assay demonstrated equal levels of sensitivity and reproducibility compared with the FDA's current validated real-time PCR assay. The assay detected three prohibited species in less than 1.5 h of total assay time, a significant improvement over the current real-time assay. These results demonstrated this assay's suitability for routine regulatory use both as a primary screening tool and as a confirmatory test.
美国食品和药物管理局（FDA）此前已经验证了一种基于实时 PCR 的检测方法，该方法目前正被 FDA 和几个州的实验室用作官方筛选方法。由于该检测方法存在几个缺陷，因此开发了一种多重实时 PCR 检测方法（MRTA），以检测三种反刍动物（牛、山羊和绵羊），该方法使用冻干珠设计。该检测方法包含两个引物或探针组：一组“反刍动物”用于检测牛、山羊和绵羊来源的材料，另一组用作内部 PCR 对照，采用冻干珠设计。该检测方法的性能是根据 FDA 兽医中心的研究办公室制定的严格验收标准进行评估的。用于检测反刍动物 DNA 的 MRTA 通过了 FDA 中心兽医办公室制定的严格验收标准，包括特异性、灵敏度和选择性。该检测方法通过检测 30 份 30 份完全用来自三种反刍动物的 0.1%（wt/wt）加工材料强化的饲料样品，满足了灵敏度和重现性要求。在整个评估期间，该检测方法对阴性对照的选择性达到 100%（0.0%的假阳性率）。该检测方法在样品选择和试剂制备方面都表现出了稳健性。第二和第三分析员的试验结果证实了书面标准操作程序的质量，并保持了结果的一致性。参与同行验证试验的外部实验室在鉴定牛骨肉粉时表现出 100%的特异性，同时假阳性率为 0.03%。与 FDA 目前验证的实时 PCR 检测方法相比，该检测方法的灵敏度和重现性水平相当。该检测方法在不到 1.5 小时的总检测时间内检测到三种违禁物种，与当前的实时检测方法相比有了显著的改进。这些结果表明，该检测方法适合常规监管使用，既可以作为初步筛选工具，也可以作为确认测试。
