首页|一种基于线粒体COX3基因的赤狐皮毛TaqMan探针荧光PCR检测方法的建立

一种基于线粒体COX3基因的赤狐皮毛TaqMan探针荧光PCR检测方法的建立

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为建立一种快速检测赤狐皮毛的方法,试验根据赤狐皮毛线粒体COX3 基因保守区设计了特异性引物和TaqMan探针,通过优化反应条件建立了一种检测赤狐皮毛的TaqMan探针荧光PCR检测方法.结果表明,该方法对赤狐皮毛具有特异性,不与兔皮毛、山羊皮毛、绵羊皮毛、貂皮毛和貉子皮毛发生交叉反应.该方法对质粒标准对照(PUC-VV-)的最适线性检测范围为4.8×101~4.8×108 copies/μL,标准曲线方程为Y =-3.4752X + 40.118,相关系数(r2)为0.9897,检测下限为48 copies/μL.对3个不同浓度的pCR-GPTV-ITR进行组内试验和组间试验,每个浓度的重复试验Ct值的变异系数均小于1.5%,具有良好的重现性.采用该方法检测2份赤狐皮毛为阳性,其他10份动物皮毛样品为阴性.本方法的建立为赤狐皮毛检测提供了一种快速准确的技术手段.
Development of a TaqMan Probe Fluorescence PCR Assay for Detection of Red Fox Fur Based on COX3 Gene
Abstrct To rapidly detect red fox fur,we conducted an experiment to design specific primers and a TaqMan-MGB probe based on the conserved region of the red fox fur mitochondrial COX3 gene.Through optimizing reaction conditions,we developed a real-time PCR detection method for red fox fur.Results show that the assay is 100%specific for the red fox fur without cross-reactions with rabbit fur,goat fur,sheep fur,mink fur and raccoon fur.The optimal linear detection range for the plasmid standard control(PUC-VV-)is 4.8×101-4.8×108 copies/μL,with a standard curve equation of Y = 3.4752X + 40.118,a correlation coefficient(r2)of 0.9897,and a detection limit of 48 copies/μL.The intra-assay and inter-assay tests were conducted for three different concentrations of pCR-GPTV-ITR.The coefficient of variation for repeated experiments at each concentration,as indicated by the Ct values,was consistently below1.5%,indicating excellent reproducibility.Application of this method for detecting two red fox fur samples yileded positive results while the other 10 animal fur samples tested negative.In conclusion,the development of this assay provides a rapid and accurate technical means for detecting red fox fur.

red fox furCOX3 genereal-time PCR

张海滨、王建华、殷其刚、肖妍、王禹、刘洋、吴冬雪、王乃福

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天津海关动植物与食品检测中心 天津 300000

赤狐皮毛 COX3基因 荧光PCR方法

天津海关科技计划项目

2022THK005

2024

中国口岸科学技术
中国质检报刊社

中国口岸科学技术

影响因子:0.051
ISSN:1002-4689
年,卷(期):2024.6(1)
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