沙门氏菌内标PCR快速检测试剂盒的研制与应用

doi:10.3864/j.issn.0578-1752.2011.16.012

摘要/Abstract

摘要： 【目的】研制一种能有效指示假阴性的PCR检测产品，用于沙门氏菌快速、准确、灵敏的检测。【方法】针对沙门氏菌invA基因、stn基因序列分别设计引物与扩增内标，建立添加扩增内标的沙门氏菌PCR检测方法，组装试剂盒，并对试剂盒的各项性能进行评价。【结果】试剂盒对147株沙门氏菌和28株非沙门氏菌的检测结果显示，所有沙门氏菌均能扩增出362 bp的目标条带，非沙门氏菌仅扩增出520 bp的内标条带。试剂盒检测沙门氏菌基因组DNA的检测限为8.0 fg/PCR，检测染菌量为4—5 CFU/10 mL的牛奶样品，增菌8—10 h即得到阳性结果。反复冻融60次或-20℃下贮存1年，均不影响试剂盒的使用效果。对食品样品的检测结果显示，阳性检出率为2/30，与国标法的检测结果相符，比未添加扩增内标的PCR检测结果（阳性检出率为1/30）准确。【结论】采用添加扩增内标的PCR试剂盒检测沙门氏菌，特异性强、灵敏度高、稳定性好，并能有效指示假阴性，提高检测的准确性，适用于食品中沙门氏菌的快速筛检。

关键词: 沙门氏菌, 聚合酶链式反应, 扩增内标, 检测试剂盒

Abstract: 【Objective】The purpose of this study was to develop a rapid and accurate detection kit for Salmonella spp. using polymerase chain reaction (PCR) with an internal amplification control (IAC). 【Method】 The specific primers and IAC were designed according to the conserved genes invA and stn in Salmonella spp. The optimization of the components and the evaluation of the parameters for the kit were carried out in this study. 【Result】 The experiment indicated that the specific 362 bp DNA fragment was amplified against 147 reference strains of Salmonella spp., while 28 strains of non-Salmonella only showed the 520 bp amplified band of IAC. The detection limit of the kit for purified genomic DNA was 8.0 fg/PCR. It was confirmed in artificial contamination assay that Salmonella spp. could be detected by the kit after 8-10 h enrichment when 4-5 CFU germs were inoculated in 10 mL milk. Thirty food samples were detected by the IAC-PCR kit in this study, and the experiments demonstrated that IAC could successfully indicate false-negative results. Besides, the kit worked well after being frozen-thawed for 60 times or stored at -20℃ for one year.【Conclusion】The IAC-PCR detection kit developed in this study has good performances in specificity, sensitivity, stability and accuracy and is suitable for the rapid and accurate detection of Salmonella spp.

Key words: Salmonella spp., PCR, IAC, detection kit

中图分类号:

T201

李小玲, 刘斌, 但现龙, 王大鹏, 周敏, 史贤明. 沙门氏菌内标PCR快速检测试剂盒的研制与应用[J]. 中国农业科学, 2011, 44(16): 3395-3402.

LI Xiao-Ling, LIU Bin, DAN Xian-Long, WANG Da-Peng, ZHOU Min, SHI Xian-Ming. Development and Application of an IAC-PCR Kit for the Rapid Detection of Salmonella spp.[J]. Scientia Agricultura Sinica, 2011, 44(16): 3395-3402.

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