D'Souza D H, Jaykus L-A
Department of Food Science, North Carolina State University, Raleigh, NC, USA.
J Appl Microbiol. 2003;95(6):1343-50. doi: 10.1046/j.1365-2672.2003.02106.x.
The purpose of this study was to apply nucleic acid sequence-based amplification (NASBA) for the detection of Salmonella enterica serovar Enteritidis (S. Enteritidis) in representative foods.
A previously reported primer and probe set based on mRNA sequences of the dnaK gene of Salmonella were used in this study. To test for possible food matrix inhibition and assay detection limits, 25-g samples of representative food commodities (fresh meats, poultry, fish, ready-to-eat salads and bakery products) were pre-enriched with and without S. Enteritidis inoculation. The NucliSens(R) Basic Kit, supplemented with enzymes from various other commercial sources, was used for RNA isolation, NASBA amplification and electrochemiluminescent (ECL) detection. The end point detection limit of the NASBA-ECL assay was equivalent to 101 CFU of S. Enteritidis per amplification reaction. When the assay was tested on noncontaminated foods, none of the food matrices produced false-positive results. Some of the food matrices inhibited the NASBA-ECL reaction unless the associated RNA was diluted 10-fold prior to amplification.
For all food items tested, positive ECL signals were achieved after 18 h of pre-enrichment and subsequent NASBA at initial inoculum levels of 102 and 101 CFU per 25 g food sample.
This rapid, semi-automated detection method has potential for use in the food, agricultural and public health sectors.
本研究旨在应用基于核酸序列的扩增技术（NASBA）检测代表性食品中的肠炎沙门氏菌肠炎血清型（肠炎沙门氏菌）。
本研究使用了先前报道的基于沙门氏菌dnaK基因mRNA序列的引物和探针组。为测试可能的食品基质抑制作用和检测限，对25克代表性食品（鲜肉、家禽、鱼类、即食沙拉和烘焙食品）样品进行接种或不接种肠炎沙门氏菌的预富集。使用补充了来自其他各种商业来源酶的NucliSens®基础试剂盒进行RNA分离、NASBA扩增和电化学发光（ECL）检测。NASBA-ECL检测的终点检测限相当于每个扩增反应10¹CFU的肠炎沙门氏菌。当该检测方法应用于未受污染的食品时，没有任何食品基质产生假阳性结果。除非相关RNA在扩增前稀释10倍，否则某些食品基质会抑制NASBA-ECL反应。
对于所有测试食品，在每25克食品样品初始接种量为10²和10¹CFU的情况下，经过18小时预富集和随后的NASBA后均获得了阳性ECL信号。
这种快速、半自动的检测方法在食品、农业和公共卫生领域具有应用潜力。
