Cloke Jonathan, Crowley Erin, Bird Patrick, Bastin Ben, Flannery Jonathan, Agin James, Goins David, Clark Dorn, Radcliff Roy, Wickstrand Nina, Kauppinen Mikko
Thermo Fisher Scientific, Wade Rd, Basingstoke, Hampshire, RG24 8PW, UK.
J AOAC Int. 2015 Sep-Oct;98(5):1301-14. doi: 10.5740/jaoacint.15-043.
The Thermo Scientific™ SureTect™ Escherichia coli O157:H7 Assay is a new real-time PCR assay which has been validated through the AOAC Research Institute (RI) Performance Tested Methods(SM) program for raw beef and produce matrixes. This validation study specifically validated the assay with 375 g 1:4 and 1:5 ratios of raw ground beef and raw beef trim in comparison to the U.S. Department of Agriculture, Food Safety Inspection Service, Microbiology Laboratory Guidebook (USDS-FSIS/MLG) reference method and 25 g bagged spinach and fresh apple juice at a ratio of 1:10, in comparison to the reference method detailed in the International Organization for Standardization 16654:2001 reference method. For raw beef matrixes, the validation of both 1:4 and 1:5 allows user flexibility with the enrichment protocol, although which of these two ratios chosen by the laboratory should be based on specific test requirements. All matrixes were analyzed by Thermo Fisher Scientific, Microbiology Division, Vantaa, Finland, and Q Laboratories Inc, Cincinnati, Ohio, in the method developer study. Two of the matrixes (raw ground beef at both 1:4 and 1:5 ratios) and bagged spinach were additionally analyzed in the AOAC-RI controlled independent laboratory study, which was conducted by Marshfield Food Safety, Marshfield, Wisconsin. Using probability of detection statistical analysis, no significant difference was demonstrated by the SureTect kit in comparison to the USDA FSIS reference method for raw beef matrixes, or with the ISO reference method for matrixes of bagged spinach and apple juice. Inclusivity and exclusivity testing was conducted with 58 E. coli O157:H7 and 54 non-E. coli O157:H7 isolates, respectively, which demonstrated that the SureTect assay was able to detect all isolates of E. coli O157:H7 analyzed. In addition, all but one of the nontarget isolates were correctly interpreted as negative by the SureTect Software. The single isolate giving a positive result was an E. coli O157:NM isolate. Nonmotile isolates of E. coli O157 have been demonstrated to still contain the H7 gene; therefore, this result is not unexpected. Robustness testing was conducted to evaluate the performance of the SureTect assay with specific deviations to the assay protocol, which were outside the recommended parameters and which are open to variation. This study demonstrated that the SureTect assay gave reliable performance. A final study to verify the shelf life of the product, under accelerated conditions was also conducted.
赛默飞世尔科技™SureTect™大肠杆菌O157:H7检测试剂盒是一种新型实时荧光定量PCR检测试剂盒，已通过美国分析化学家协会（AOAC）研究机构（RI）的性能验证方法（SM）计划，可用于生牛肉和农产品基质检测。该验证研究具体针对375克1:4和1:5比例的绞碎生牛肉及生牛肉切块，与美国农业部食品安全检验局微生物实验室指南手册（USDS-FSIS/MLG）参考方法进行对比验证；同时针对25克袋装菠菜和鲜榨苹果汁，按1:10的比例与国际标准化组织16654:2001参考方法中详述的参考方法进行对比验证。对于生牛肉基质，1:4和1:5比例的验证使得用户在富集方案上具有灵活性，不过实验室选择这两种比例中的哪一种应基于具体的检测要求。在方法开发者研究中，所有基质均由芬兰万塔的赛默飞世尔科技微生物部门以及俄亥俄州辛辛那提的Q实验室公司进行分析。其中两种基质（1:4和1:5比例的绞碎生牛肉）和袋装菠菜在AOAC-RI控制的独立实验室研究中进行了额外分析，该研究由威斯康星州马什菲尔德的马什菲尔德食品安全公司开展。通过检测概率统计分析表明，与USDA FSIS生牛肉基质参考方法相比，SureTect试剂盒无显著差异；与袋装菠菜和苹果汁基质的ISO参考方法相比，也无显著差异。分别使用58株大肠杆菌O157:H7和54株非大肠杆菌O157:H7分离株进行了包容性和排他性测试，结果表明SureTect检测试剂盒能够检测所有分析的大肠杆菌O157:H7分离株。此外，除一株外，所有非目标分离株经SureTect软件正确判定为阴性。给出阳性结果的单一分离株是一株大肠杆菌O157:NM分离株。已证实大肠杆菌O157的非运动性分离株仍含有H7基因；因此，这一结果并不意外。进行了稳健性测试，以评估SureTect检测试剂盒在检测方案存在特定偏差时的性能，这些偏差超出了推荐参数范围且可能存在变化。该研究表明SureTect检测试剂盒性能可靠。还进行了一项最终研究，在加速条件下验证产品的保质期。
