实时荧光 PCR 和双标记荧光共振能量转移探针熔解峰分析用于检测牛肉产品中大肠杆菌 O157:H7 的比较性能评估。

Comparative Performance Evaluation of Real-Time PCR and Dual-Labeled Fluorescence Resonance Energy Transfer Probe-Based Melt Peak Analysis for the Detection of Escherichia coli O157:H7 in Beef Products.

机构信息

1 U.S. Department of Agriculture, Agricultural Research Service, U.S. Meat Animal Research Center, Spur 18D, Clay Center, Nebraska 68933, USA.

2 bioMérieux, Inc., 595 Anglum Road, Hazelwood, Missouri 63042, USA.

出版信息

J Food Prot. 2019 Mar;82(3):507-512. doi: 10.4315/0362-028X.JFP-18-366.

DOI:10.4315/0362-028X.JFP-18-366

PMID:30810380

Abstract

Contaminated beef and beef products remain a frequent vehicle for the transmission of Escherichia coli O157:H7. The current U.S. Department of Agriculture (USDA) Food Safety and Inspection Service (FSIS) regulatory testing for E. coli O157:H7 uses the method described in the USDA-FSIS Microbiology Laboratory Guidebook (MLG), chapter 5. At times, described presumptive test results are nonconfirmable, suggesting that recent PCR technological advancements and presumed enhanced sensitivity and specificity may offer beneficial changes. Here, we have evaluated the precision and sensitivity of a fluorescence resonance energy transfer-based real-time PCR assay called ECO for the detection of E. coli O157:H7. ECO detects the gene target specific to both E. coli O157:H7 and E. coli O157:non-H7 but distinguishes the two by using a melt curve analysis. A total of 3,113 O157:H7 and O157:non-H7 isolates were used to define this melting temperature-based criteria. The simulated comparative performance evaluation in the spiked beef samples indicated detection of 3 of 3 samples by ECO at <3.3 log CFU/mL, whereas MLG only detected 1 of 3 (<3.3 log CFU/mL). Using modified tryptic soy broth-enriched natural beef and veal product samples ( n = 452), the comparative sensitivity, specificity, false-positive rate, and false-negative rate against culture between MLG and ECO were 75 versus 92%, 91 versus 99%, 8.9 versus 0.77%, and 25 versus 8.3%, respectively. Positive predictive value, negative predictive value, and the overall accuracy were found to be 56 versus 94%, 96 versus 98%, and 88 versus 98%, for MLG and ECO, respectively. These data demonstrate that the ECO assay is comparable to MLG detection of E. coli O157:H7 and offers improved sensitivity.

摘要

受污染的牛肉和牛肉产品仍然是大肠杆菌 O157:H7 传播的常见媒介。目前美国农业部（USDA）食品安全检验局（FSIS）对大肠杆菌 O157:H7 的监管检测使用 USDA-FSIS 微生物实验室指南（MLG）第五章中描述的方法。有时，描述的初步检测结果不可确认，这表明最近的 PCR 技术进步和假定的更高灵敏度和特异性可能会带来有益的改变。在这里，我们评估了一种称为 ECO 的基于荧光共振能量转移的实时 PCR 检测方法检测大肠杆菌 O157:H7 的精密度和灵敏度。ECO 检测到特定于大肠杆菌 O157:H7 和大肠杆菌 O157:非 H7 的基因靶标，但通过使用熔解曲线分析来区分两者。总共使用了 3113 个 O157:H7 和 O157:非 H7 分离株来定义基于这种熔点的标准。在添加牛肉样品中的模拟比较性能评估表明，ECO 在 <3.3 log CFU/mL 时可检测到 3 个样品中的 3 个，而 MLG 仅检测到 1 个样品 (<3.3 log CFU/mL)。使用改良的胰蛋白酶大豆肉汤富集天然牛肉和小牛肉产品样品（n=452），与 MLG 相比，ECO 的比较灵敏度、特异性、假阳性率和假阴性率分别为 75%对 92%、91%对 99%、8.9%对 0.77%和 25%对 8.3%。MLG 和 ECO 的阳性预测值、阴性预测值和总准确性分别为 56%对 94%、96%对 98%和 88%对 98%。这些数据表明，ECO 检测方法与 MLG 检测大肠杆菌 O157:H7 相当，并且具有更高的灵敏度。