摘要
针对我国缺乏适用于创伤弧菌检测的质粒标准样品的现状,本研究开展了创伤弧菌鉴定即用型定性质粒标准样品的研制和应用工作.首先构建了创伤弧菌毒力基因vvhA的重组质粒,经测序验证后制备成质粒标准样品冻干粉,然后对其进行PCR定性检测及紫外分光光度计法定量分析.均匀性检验结果表明,样品间无显著差异,均匀性良好,符合预期目标;短期稳定性检验结果表明,样品能在 4℃、37℃条件下稳定保存 14 天;长期稳定性检验结果表明,样品能在-20℃条件下稳定保存至少 12 个月.研究结果表明,创伤弧菌质粒定性标准样品的均匀性和稳定性均符合国家定性标准样品的要求,为创伤弧菌的快速、高通量的定性鉴定分析提供了可靠的参考物质.将标准样品应用于鱼类、贝类等 10 份海鲜类食品样品的检测中,经传统培养法验证,检测结果准确无误.本研究所研制的创伤弧菌质粒定性标准样品具有较好的商业应用潜力,为其在食品检测领域的推广应用奠定了重要基础.
Abstract
Due to lack of plasmid standard sample suitable for detection of Vibrio vulnificus in China,a ready-to-use certified plasmid reference material was developed herein.The recombinant plasmids of vvhA gene of V.vulnificus was constructed firstly and then their freeze-dried plasmid powders were prepared after sequen-cing verification and being detected by PCR and UV spectrophotometry.The uniformity test showed no signifi-cant difference between samples,and the uniformity was as expected.The stability test showed that the pre-pared plasmid samples could be stably preserved for 14 days at 4℃or 37℃,and for at least 12 months at-20℃.The overall results indicated that the uniformity and stability of the vvhA gene recombinant plasmid qualitative standard samples could meet the requirements of national qualitative standard samples,which pro-vided reliable reference material for rapid and high-throughput qualitative identification of V.vulnificus.The samples were used as positive control in the detection of 10 seafood samples such as fish and shellfish.and the results were confirmed to be accurate by traditional culture method.Above all,the qualitative standard samples of vvhA gene recombinant plasmid developed in this study had great potential in commercial application,laying an important foundation for its application in food detection.
基金项目
国家重点研发计划"食品安全关键技术研发"重点专项(2017YFC1601400)
山东省重点研发计划项目(2022TZXD0022)
泰山学者工程专项(tsqn201909168)
山东省自然科学基金青年基金项目(ZR2020QC226)
济南市"新高校 20 条"项目(202228062)
山东省农业科学院国际科技合作专项(CXGC2022F09)
维普
万方数据