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中国生物工程杂志

China Biotechnology
China Biotechnology
China Biotechnology  2017, Vol. 37 Issue (12): 67-76    DOI: 10.13523/j.cb.20171210
Orginal Article     
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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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     
Received: 26 May 2017      Published: 16 December 2017
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Yan-yan LIU
Hui-rong LI
Yue HU
Yang-yang FAN
Xiang-ming LI
Qing-qing TAN
Jia-qiang WU
Xun BU
Cite this article:

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.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.20171210     OR     https://manu60.magtech.com.cn/biotech/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
Table 1 Primer and proble sequence of fox, mink, racoon and dog
Fig.1 Sequence results of fox, mink, raccoon and dog
Fig.2 Primer concentration detection of fox, mink, raccoon and dog
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
Table 2 Fox, mink, raccoon and dog multiple real-time PCR system
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
Table 3 Primer and proble specificity detection of fox, mink, raccoon and dog
Fig.3 Specific amplification curve of fox, mink, raccoon ,dog and negative control
Fig.4 The sensitivity detection of fox, mink, raccoon and dog
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
Table 4 Test results of fox, mink, raccoon and dog derived materials in the commercial meat products
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