实时荧光PCR技术检测4种常见高组胺鱼成分方法的建立及初步应用  

Establishment and Preliminary Application of Real-time PCR for Detecting Four Common Histamine-rich Fish Ingredient

作　　者：刘立兵 周藏 付琦 孙晓霞 王金凤 钱云开 艾连峰 王建昌 LIU Li-Bing;ZHOU Cang;FU Qi;SUN Xiao-Xia;WANG Jin-Feng;QIAN Yun-Kai;AI Lian-Feng;WANG Jian-Chang(Technology Center of Shijiazhuang Customs,Shijiazhuang 050051;School of Public Health,Hebei Medical University,Shijiazhuang 050017;Technology Center of Qinhuangdao Customs,Qinhuangdao 066004)

机构地区：[1]石家庄海关技术中心,石家庄050051 [2]河北医科大学公共卫生学院,石家庄050017 [3]秦皇岛海关技术中心,秦皇岛066004

出　　处：《中国口岸科学技术》2023年第3期50-58,共9页China Port Science and Technology

基　　金：国家重点研发计划“科技冬奥”重点专项(2020YFF0305004);河北省省级科技计划资助项目(21375501D)。

摘　　要：为建立针对常见高组胺鱼成分的实时荧光PCR(real-time PCR)检测方法,本研究以日本鲭的ATP-6基因、金枪鱼的BGH基因、秋刀鱼和沙丁鱼的Cytb基因为靶基因设计特异性的引物和探针,并通过反应体系的优化试验、特异性试验和灵敏性试验,建立了针对4种高组胺鱼成分的实时荧光PCR检测方法。结果显示,该方法特异性强,对其他常见鱼类物种均无特异性扩增,日本鲭、沙丁鱼、秋刀鱼和金枪鱼的灵敏性可分别达到10.0 pg/μL、1.0 pg/μL、1.0 pg/μL、1.0 pg/μL;以人工模拟样品进行检出限分析,建立的实时荧光PCR方法的检出限均可达0.1%;对市售的36份鱼肉制品进行检测,日本鲭、沙丁鱼、秋刀鱼和金枪鱼成分的检出率分别为11.11%(4/36)、5.56%(2/36)、5.56%(2/36)和16.67%(6/36)。本研究建立的针对4种常见高组胺鱼成分的实时荧光PCR检测方法,操作简便、结果可靠,可为我国鱼肉制品进出口检验监管和食品安全监管提供技术支撑。In order to establish a real-time PCR method for the detection of common histamine-rich fish ingredient,the ATP-6 gene of Scomber japonicus,the BGH gene of tuna fish,the Cytb gene of saury and sardine were selected as target genes,and the specific primers and probes were designed based on the above targets.Through the optimization of reaction system,a real-time PCR assay for four histamine-rich fish ingredient was developed and the specificity and sensitivity analysis were performed.The results showed that the developed real-time PCR assay was highly specific,with no cross reactions with other common fish species.The assay also showed high sensitivity with the limit of detection for Scomber japonicus,saury,sardine and tuna fish derived ingredients of 10.0 pg/μL,1.0 pg/μL,1.0 pg/μL and 1.0 pg/μL,respectively.For the artificially simulated samples,the established real-time PCR assay could detect as low as of 0.1%target species in the mixed samples.Furthermore,36 different fish products were tested by the real-time PCR assay and the detection rates were 11.11%(4/36),5.56%(2/36),5.56%(2/36)and 16.67%(6/36)for Scomber japonicus,saury,sardine and tuna fish ingredients,respectively.The real-time PCR assay for four common histaminerich fish ingredient developed in this study was simple and reliable,which can provide technical supports for the import and export inspection and food safety control of fish products in China.

关 键 词：高组胺鱼 实时荧光PCR 鱼肉制品 食品安全 

分 类 号：TS254.7[轻工技术与工程—水产品加工及贮藏工程] O657.3[轻工技术与工程—食品科学与工程]