Caballero Oscar, Alles Susan, Le Quynh-Nhi, Gray R Lucas, Hosking Edan, Pinkava Lisa, Norton Paul, Tolan Jerry, Mozola Mark, Rice Jennifer, Chen Yi, Ryser Elliot, Odumeru Joseph
Neogen Corp., 620 Lesher Pl, Lansing, MI 48912, USA.
J AOAC Int. 2016 Jan-Feb;99(1):112-23. doi: 10.5740/jaoacint.15-0200. Epub 2016 Jan 29.
Work was conducted to validate performance of the ANSR(®) for Listeria monocytogenes method in selected food and environmental matrixes. This DNA-based assay involves amplification of nucleic acid via an isothermal reaction based on nicking enzyme amplification technology. Following single-step sample enrichment for 16-24 h for most matrixes, the assay is completed in 40 min using only simple instrumentation. When 50 distinct strains of L. monocytogenes were tested for inclusivity, 48 produced positive results, the exceptions being two strains confirmed by PCR to lack the assay target gene. Forty-seven nontarget strains (30 species), including multiple non-monocytogenes Listeria species as well as non-Listeria, Gram-positive bacteria, were tested, and all generated negative ANSR assay results. Performance of the ANSR method was compared with that of the U.S. Department of Agriculture, Food Safety and Inspection Service Microbiology Laboratory Guidebook reference culture procedure for detection of L. monocytogenes in hot dogs, pasteurized liquid egg, and sponge samples taken from an inoculated stainless steel surface. In addition, ANSR performance was measured against the U.S. Food and Drug Administration Bacteriological Analytical Manual reference method for detection of L. monocytogenes in Mexican-style cheese, cantaloupe, sprout irrigation water, and guacamole. With the single exception of pasteurized liquid egg at 16 h, ANSR method performance as quantified by the number of positives obtained was not statistically different from that of the reference methods. Robustness trials demonstrated that deliberate introduction of small deviations to the normal assay parameters did not affect ANSR method performance. Results of accelerated stability testing conducted using two manufactured lots of reagents predicts stability at the specified storage temperature of 4°C of more than 1 year.
开展了相关工作，以验证ANSR（®）用于检测单核细胞增生李斯特菌的方法在选定食品和环境基质中的性能。这种基于DNA的检测方法涉及通过基于切口酶扩增技术的等温反应来扩增核酸。对于大多数基质，经过16 - 24小时的单步样品富集后，仅使用简单仪器即可在40分钟内完成检测。在对50株不同的单核细胞增生李斯特菌菌株进行包容性测试时，48株产生了阳性结果，例外的是经PCR确认缺少检测靶基因的两株菌株。对47株非靶标菌株（30个物种）进行了测试，包括多种非单核细胞增生李斯特菌的李斯特菌属物种以及非李斯特菌属的革兰氏阳性菌，所有这些菌株的ANSR检测结果均为阴性。将ANSR方法的性能与美国农业部食品安全与检验局微生物实验室指南中用于检测热狗、巴氏杀菌液态蛋以及从接种的不锈钢表面采集的海绵样品中单核细胞增生李斯特菌的参考培养程序进行了比较。此外，还根据美国食品药品监督管理局细菌学分析手册中用于检测墨西哥式奶酪、哈密瓜、豆芽灌溉水和鳄梨酱中单核细胞增生李斯特菌的参考方法来衡量ANSR的性能。除了16小时的巴氏杀菌液态蛋这一唯一例外情况外，通过获得的阳性数量量化的ANSR方法性能与参考方法在统计学上没有差异。稳健性试验表明，故意对正常检测参数引入小偏差不会影响ANSR方法的性能。使用两批生产的试剂进行的加速稳定性测试结果预测，在规定的4°C储存温度下，稳定性超过1年。
