基于全自动核酸提取的荧光定量PCR鉴别羊肉制品真伪  

Identification of mutton products by flourescence quationtitative PCR based on automatic nucleic acid extration

作　　者：柳梦思 时国强 卢鑫 张伟[2] 石磊 LIU Meng-Si;SHI Guo-Qiang;LU Xin;ZHANG Wei;SHI Lei(Hebei Sanshibio-tech Co.,Ltd.,Shijiazhuang 050035,China;College of Life Sciences,Hebei Agricultural University,Baoding 071000,China)

机构地区：[1]河北三狮生物科技有限公司,石家庄050035 [2]河北农业大学生命科学学院,保定071000

出　　处：《食品安全质量检测学报》2022年第10期3148-3155,共8页Journal of Food Safety and Quality

基　　金：石家庄高新区产学研合作项目(20430007C)。

摘　　要：目的建立一种基于自动化的快速核酸提取方法,联合三重实时荧光定量PCR技术,鉴别羊肉制品的真伪。方法分别采用全自动核酸提取法、磁珠手提法与柱提法对不同状态羊肉制品进行核酸提取,比较不同方法的提取时间差异,通过三重实时荧光定量PCR扩增结果评估不同方法的提取效果。以GB/T38164—2019《常见畜禽动物源性成分检测方法实时荧光PCR法》为对照,对市售的羊肉制品进行平行检测,评估本方法的检测性能。结果与磁珠手提法、柱提法相比,全自动核酸提取法的提取时间缩短了15~30 min,且提取的核酸扩增效果更好。全自动核酸提取法在羊猪混合样品中羊源核酸检出限为0.010mg/g。全自动核酸提取法在生鲜肉、生肉及熟肉制品等多种类型产品中提取的羊源核酸PCR检测结果的变异系数(coefficient of variation,CV)均小于3.000%。分别采用三重实时荧光定量PCR法与GB/T38164—2019对全自动提取的21份市售的涵盖不同类型的羊肉制品核酸进行平行检测,符合率为100.00%。结论本研究方法可为羊肉制品掺假的快速真伪鉴别提供操作简便、稳定灵敏、快速准确的整套解决方案。Objective To establish a rapid nucleic acid extraction method based on automation,combined with triple real-time fluorescence quantitative PCR technology,to identify the authenticity of mutton products.Methods Automatic nucleic acid extraction method,magnetic bead hand-held method and column extraction method were used to extract nucleic acid from mutton products in different states.The extraction time differences of different methods were compared,and the extraction effects of different methods were evaluated by triple real-time fluorescence quantitative PCR amplification results.Taking the GB/T 38164—2019 Common methods for determination of components of livestock,poultry and animal origin-Real-time fluorescence PCR method as a control,parallel detection of commercially available mutton products was carried out to evaluate the detection performance of this method.Results Compared with the magnetic bead method and the column extraction method,the extraction time of the automatic nucleic acid extraction method was shortened by 15-30 min,and the extracted nucleic acid amplification effect was better.The detection limit of sheep-derived nucleic acid in the mixed samples of sheep and pigs by the automatic nucleic acid extraction method was 0.010 mg/g.The coefficient of variation(CV)of the PCR detection results of sheep-derived nucleic acid extracted from various types of products such as raw fresh meat,raw meat and cooked meat products by the automatic nucleic acid extraction method were all less than 3.000%.The triple real-time fluorescence quantitative PCR method and GB/T 38164—2019 were used for parallel detection of 21 kinds of commercially available nucleic acids covering different types of mutton products extracted by automatic extraction,and the coincidence rate was 100.00%.Conclusion This research method can provide a complete solution that is easy to operate,stable,sensitive,fast and accurate for the rapid authenticity identification of adulterated mutton products.

关 键 词：全自动核酸提取 磁珠法 羊肉 真伪鉴别 三重实时荧光定量PCR 

分 类 号：TS251.53[轻工技术与工程—农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]