基于分子信标技术的肉类食品动物源性成分多重筛检方法  

Multiple Identification Method of Animal Species in Meat Food Based on Molecular Beacon Technology

作　　者：承海 朱洪亮 姚振明 毛玲燕 张胜男 王慧君 邢家溧 Cheng Hai;Zhu Hongliang;Yao Zhenming;Mao Lingyan;Zhang Shengnan;Wang Huijun;Xing Jiali(Ningbo Academy of Product and Food Quality Inspection(Ningbo Fibre Inspection Institute),Ningbo 315408,Zhejiang;Jiaxing Institute for Food,Drug and Product Quality Control,Jiaxing 330400,Zhejiang)

机构地区：[1]宁波市产品食品质量检验研究院(宁波市纤维检验所),浙江宁波315408 [2]嘉兴市食品药品与产品质量检验检测院,浙江嘉兴330400

出　　处：《中国食品学报》2023年第6期378-384,共7页Journal of Chinese Institute Of Food Science and Technology

基　　金：浙江省市场监管局雏鹰计划项目(CY2023322);宁波市泛3315创新团队项目(2018B-18-C);宁波国家高新精英项目(甬高科[2018]63号)。

摘　　要：建立一种基于通用型分子信标检测及探针熔解曲线分析技术的肉类食品动物源性成分多重筛检方法。通过对猪、牛、羊等9种常见食用畜禽动物基因组多序列比对及生物信息学分析,选取线粒体基因组16s r DNA中具有特定结构的基因片段作为检测靶序列,设计单一的通用型引物和长链分子信标。以标准基因重组载体及实体样品基因组为检测对象,确定各动物源性成分特征退火温度(Tm),建立基于实时荧光PCR及熔解曲线分析的标准检测方法,并对筛检方法的灵敏度和特异性进行考察。应用所建立检测方法,9种畜禽动物基因组扩增产物单一,测序结果与参考序列一致。对梯度稀释的猪标准基因重组载体检测发现,终浓度10~8~10~1copies/μL模板扩增Ct值范围为15~36个循环(回归系数R~2=0.99),熔解峰特征Tm为(68.0±0.1)℃。对9类畜禽肉类标准基因重组载体和实体样品进行独立检测,各类动物源性基因均形成可辨识的特异性熔解峰(Tm分别为猪68.0℃、牛64.3℃、羊65.1℃、驴65.8℃、马63.4℃、驼60.6℃、狗61.8℃、鸡52.6℃、鸭56.7℃),混合样品检测可见独立的种属特征熔解峰或融合形成新熔解峰。基于通用型引物及分子信标的实时荧光PCR检测方法具有理想的灵敏度和特异性,可实现对常见畜禽肉类食品源性成分的单管多重鉴定,在肉类食品源性成分筛检方面具有一定的推广应用价值。To establish a multiple identification method of animal species in meat by using a universal molecular beacon with probe melting curve analysis.Through multi sequence alignment of the genomes of 9 common edible livestock and poultry,the gene fragments with specific structure in the 16S rDNA of the mitochondrial genome were selected as the detection target sequence.A pair of primers and a long-chain molecular beacon for universal detection were designed by the method of bioinformatics analysis.The characteristic annealing temperature(Tm)of different animal derived components was determined based on real-time polymerase chain reaction(RT-PCR)and melting curve analysis,while the sensitivity and specificity of the identification method were investigated.The RT-PCR based on universal primers and molecular beacon was performed on genome samples derived from 9 animal species.The amplified product of each sample was single,and the sequencing result was consistent with its reference sequence.While a dilution range of 108 to 101 copies/μL of standard DNA of pork was used as template,the RT-PCR was performed validly with incremental Ct value from 15 to 36 cycles,and the characteristic Tm was about(68.0±0.1)℃steadily by melting curve analysis.The detection result also showed that identifiable specific annealing temperature was obtained for each sample derived from the livestock and poultry meat investigated with Tm of 68.0℃for pigs,64.3℃for cattles,65.1℃for sheep,65.8℃for donkeys,63.4℃for horses,60.6℃for camels,61.8℃for dogs,52.6℃for chickens and 56.7℃for ducks.While the mixed genome derived from different animal species was detected,the results could show the independent melting peaks of various species genes or the emergence of a new fusion peak.The RT-PCR detection method based on universal primers and molecular beacon can realize the single-tube multiple identification of common livestock and poultry meat food species with ideal sensitivity and specificity.With high application value,this method cou

关 键 词：分子信标 肉类食品 熔解曲线 鉴定 

分 类 号：TS251.7[轻工技术与工程—农产品加工及贮藏工程]