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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2017, Vol. 37 Issue (12): 67-76    DOI: 10.13523/j.cb.20171210
技术与方法     
饲料中狐狸、水貂、貉子和狗源性的五重实时荧光PCR检测方法的建立 *
刘艳艳1,李会荣2,胡悦1,范阳阳1,李祥明2,谭晴晴1,吴家强3,步迅1*()
1 山东省农业科学院生物技术研究中心 济南 250100
2 山东省饲畜牧兽医局饲料质量检验所 济南 250022
3 山东省农业科学院家禽所 济南 250100
Multiplex Fluorescent Real-time PCR Detection of Fox, Mink, Raccoon and Dog Derived Materials in Feedstuff
Yan-yan LIU1,Hui-rong LI2,Yue HU1,Yang-yang FAN1,Xiang-ming LI2,Qing-qing TAN1,Jia-qiang WU3,Xun BU1*()
1 Biotechnology Research Center Shandong Academy of Agricultural Sciences, Jinan 250100, China
2 Feed Quality Inspection Institute of Shandong Animal Husbandry and Veterinary Bureau, Jinan 250022, China
3 Poultry Institute of Shandong Academy of Agricultural Sciences, Jinan 250100, China
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摘要：

为了快速鉴别饲料中的狐狸、水貂、貉子和狗源性成分,根据线粒体16S rDNA种间保守序列,设计合成针对狐狸、水貂、貉子和狗的特异性引物和探针,通过对荧光PCR反应体系和反应条件的优化筛选,建立了多重实时荧光PCR方法,在同一PCR反应体系中可以同时完成4种动物源性成分检测。通过对15种其他物种的源性成分的检测,结果表明所设计的引物和探针具有很好的物种特异性,且灵敏度高,狐狸、水貂、貉子和狗的DNA检出限为0.01ng。对40份样品检测,其中5份检测出貉子、狐狸和水貂源性成分。结果表明,该方法可以有效地鉴别出饲料中狐狸、水貂、貉子和狗源性成分,同时适用于相关动物产品中。
关键词： 线粒体16S;  rDNA;  多重实时荧光PCR;  狐狸、水貂、貉子和狗动物源性成分    
Abstract:

In order to identify the fox, mink, raccoon and dog-derived components in feed, the specific primers and probes for fox, mink, raccoon and dog were designed and synthesized according to the conserved sequence of mitochondrial 16S rDNA. Fluorescent PCR reaction system and reaction conditions were optimized. A number of real-time fluorescent PCR methods were established. Four kinds of animal-derived components were detected in the same PCR reaction system. The developed method could successfully detect four kinds of animal derived materials including fox, mink, raccoon and dog in the same reaction. Through the detection of the source components of 15 other species, the results showed that the primers and probes designed in this experiment had good species specificity. The method has high sensitivity, fox, mink, raccoon and dog DNA detection limit of 0.01ng. Of the 40 samples tested, 5 of them detected raccoon, fox and mink-derived ingredients. The results showed that the method has high specificity and sensitivity. It could effectively identify the fox, mink, raccoon and dog - derived components in the feed, and it was suitable for the related animal products.
Key words: Mitochondrial 16S    rRNA    Multiple real-time fluorescent PCR    Foxmink raccoon and dog derived materials
收稿日期: 2017-05-26 出版日期: 2017-12-16
ZTFLH:  Q819  
基金资助: 国家自然基金青年科学基金(31702215)、泰山学者项目,山东省现代农业产业技术体系(SDAIT)资助项目
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刘艳艳
李会荣
胡悦
范阳阳
李祥明
谭晴晴
吴家强
步迅

引用本文:

刘艳艳,李会荣,胡悦,范阳阳,李祥明,谭晴晴,吴家强,步迅. 饲料中狐狸、水貂、貉子和狗源性的五重实时荧光PCR检测方法的建立 *[J]. 中国生物工程杂志, 2017, 37(12): 67-76.

Yan-yan LIU,Hui-rong LI,Yue HU,Yang-yang FAN,Xiang-ming LI,Qing-qing TAN,Jia-qiang WU,Xun BU. Multiplex Fluorescent Real-time PCR Detection of Fox, Mink, Raccoon and Dog Derived Materials in Feedstuff. China Biotechnology, 2017, 37(12): 67-76.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20171210    或    https://manu60.magtech.com.cn/biotech/CN/Y2017/V37/I12/67

引物名称 引物与探针序列 扩增长度(bp)
线粒体16S rRNA基因通用引物 上游引物:MtUF: 5' CTTCCCGTGAAGAGGCGGGAATAC 3'
下游引物:MtUR: 5' TCCGAGGTCACCCCAACCTAAA 3'		 152
貉子特异探针(NYP) 5' JOE-CTTTAATTACTTAACCCAAATTTATGGCCAA-BHQ2 3'
水貂特异探针(SD-P) 5' CY3-CCCATAATAATTTATAAACTCACCTACCAGGTCTAA-BHQ2 3'
狐狸特异探针(H-P) 5' FAM-TTAGCCCAAACCCATGAAATCCAAACCCCT-BHQ1 3'
狗特异探针(CLF) 5' ROX-CTAACCCAAACTTATGGATACTAGATACCTACA-BHQ2 3'
内标探针(IMP) 5'CY5-AAGTACGCTCCATTGGTGACCTCA-BHQ2 3'
内标序列 CTTCCCGTGAAGAGGCGGGAATACAGCACGCCGTAAGCTTAAC
CTGACGCTAGTAGGCAAGTACGCTCCATTGGTGACCTCATTTTTT
AGGTTGGGGTGACCTCGGA		 107
表1  狐狸、水貂、貉子和狗源性成分的引物与探针序列
图1  狐狸、水貂、貉子和狗测序结果图
图2  狐狸、水貂、貉子和狗引物浓度优化实验
试剂名称 组分名称 加量(μl) 终浓度
2×TaqManMaster Mix 10 1×
10× Primer Mix MtUF F/ MtUF R 2 0. 50μmol/L
10× Probe Mix 水貂
SD-P(CY3) 0.25μmol/L
狐狸
H-P(FAM) 0.025μmol/L
貉子 2 0.025μmol/L
NYP-(JOE)
狗 0.10μmol/L
CLF(ROX)
内标(CY5) 0.025μmol/L
内标质粒 1 1pg/μl
DNA模板 2 1~50ng/μl
ddH2O 3
表2  狐狸、水貂、貉子和狗多重实时荧光PCR体系
物种 荧光定量PCR检测Ct值
狐狸(FAM) 貉子(JOE) 水貂(CY3) 狗(ROX) 内标(CY5)
貉子 N 24.62+0.05 N N 31.26+0.02
狐狸 28.35±0.02 N N N 30.59±0.05
水貂 N N 28.21+0.04 N 29.56+0.05
狗 N N N 27.59+0.04 29.42+0.04
牛 N N N N 27.60+0.02
绵羊 N N N N 28.89+0.04
山羊 N N N N 27.21+0.04
猪 N N N N 26.32+0.04
兔 N N N N 27.49+0.04
鸭 N N N N 23.14+0.04
鸡 N N N N 27.28+0.04
鹅 N N N N 24.21+0.04
鱼 N N N N 26.96+0.04
玉米 N N N N 26.53+0.04
小麦 N N N N 25.32+0.04
表3  狐狸、水貂、貉子和狗的引物及探针特异性试验
图3  狐狸、水貂、貉子、狗和阴性对照特异性扩增曲线图
图4  狐狸、水貂、貉子和狗灵敏度扩增曲线图
样品名称 荧光定量PCR检测Ct值 结结果显示
狐狸(FAM) 貉子(JOE) 水貂(CY3) 狗(ROX) 内标(CY5)
鸡肉粉1 N 26.23+0.05 N N 30.26+0.02 检出貉源性成分
鸡肉粉2 N N N N 28.59+0.05 未检出
鸡肉粉3 26.23+0.05 N 29.21+0.04 N 29.56+0.02 检出狐狸和水貂源性成分
鸡肉粉4 N N N N 29.42+0.05 未检出
鸡肉粉5 N N N N 27.60+0.03 未检出
猪肉粉1 27.89 N N N 28.89+0.04 检出狐狸源性成分
猪水解蛋白粉1 N N N N 27.21+0.04 未检出
鸭粉 25.47 N N N 26.32+0.04 检出狐狸源性成分
鸭粉2 N N N N 27.49+0.04 未检出
鸭粉3 N N N N 23.14+0.04 未检出
鱼粉1 N N N N 27.28+0.04 未检出
鱼粉2 N N N N 24.21+0.04 未检出
鱼粉3 29.32 N N N 26.96+0.04 检出狐狸源性成分
猪水解蛋白粉2 N N N N 28.53+0.04 未检出
猪肉粉2 N N N N 29.32+0.04 未检出
猪肉粉3 N N N N 27.53+0.04 未检出
猪肉粉4 N N N N 25.32+0.04 未检出
猪肉粉5 N N N N 25.83+0.04 未检出
猪肉粉6 N N N N 30.32+0.04 未检出
猪肉粉7 N N N N 29.49+0.04 未检出
猪肉粉8 N N N N 27.62+0.04 未检出
表4  市售肉制品中狐狸、貉子、水貂和狗源性成分检测
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