U.S. Food and Drug Administration, Center for Veterinary Medicine, Office of Research, Laurel, Maryland 20708, USA.
J Food Prot. 2009 Nov;72(11):2368-74. doi: 10.4315/0362-028x-72.11.2368.
Four real-time PCR assays that can be used with U.S.- and European Union-rendered materials to detect three ruminant species (bovine, caprine, and ovine) and a select set of avians (chicken, goose, and turkey) were developed. This method was evaluated against stringent acceptance criteria previously developed by the U.S. Food and Drug Administration, Center for Veterinary Medicine's Office of Research. Acceptance criteria for determining success used a statistical approach requiring a 90% probability of achieving the correct response, within a 95% confidence interval. A minimum detection level of 0.1% meat and bone meal (MBM) was required, consistent with the sensitivity of the validated PCR-based method currently used by the U.S. Food and Drug Administration as an aid in enforcement of the Agency's feed ban. PCR primer specificity was determined by using a panel of DNA samples derived from 16 different animal species. The method is able to detect 0.1% rendered material in complete feed in less than 1.5 h of total assay time, a significant improvement over the current method, which requires 7 to 8 h for completion. The real-time assay for the detection of animal material passed stringent acceptance criteria for sensitivity, selectivity, and specificity. The method also passed ruggedness, real-time platform, and second analyst trials. Two external laboratories participating in a peer-verification trial demonstrated 100% specificity in identifying bovine MBM, ovine MBM, or caprine meat meal, while exhibiting a 0.6% rate of false positives. These results demonstrated that this method was capable of being used by other laboratories.
开发了四种实时 PCR 检测方法，可用于检测美国和欧盟处理材料中的三种反刍动物（牛、山羊和绵羊）和一组选定的禽类（鸡、鹅和火鸡）。该方法根据美国食品和药物管理局兽医研究办公室之前制定的严格验收标准进行了评估。用于确定成功的验收标准采用了一种统计方法，要求在 95%置信区间内达到正确响应的概率为 90%。要求最低检测水平为 0.1%肉骨粉（MBM），与目前美国食品和药物管理局使用的基于 PCR 的验证方法的灵敏度一致，该方法是作为执法机构饲料禁令的辅助手段。通过使用源自 16 种不同动物物种的 DNA 样本面板来确定 PCR 引物的特异性。该方法能够在不到 1.5 小时的总检测时间内检测到 0.1%的完全饲料中的加工材料，这比目前需要 7 到 8 小时才能完成的方法有了显著的改进。用于检测动物材料的实时检测方法通过了灵敏度、选择性和特异性的严格验收标准。该方法还通过了耐用性、实时平台和第二分析师试验。参加同行验证试验的两个外部实验室在鉴定牛 MBM、绵羊 MBM 或山羊肉粉时表现出 100%的特异性，而假阳性率为 0.6%。这些结果表明，该方法能够被其他实验室使用。
