利用TaqMan微流控阵列同步检测多种动物源性成分

doi:10.3785/j.issn.1008-9209.2019.04.221

浙江大学学报（农业与生命科学版）

2020, Vol. 46

Issue (2): 189-200 DOI: 10.3785/j.issn.1008-9209.2019.04.221

食品科学

利用TaqMan微流控阵列同步检测多种动物源性成分

虞惠贞¹(

),侯寒黎²,尹文秀¹,张荃¹,孙超¹,张明哲¹,张晓峰¹,苗丽³,吴姗¹(

)

^1.浙江省检验检疫科学技术研究院，杭州 310012
^2.钱江海关，杭州 310007
^3.郑州海关技术中心，郑州 450003

Application of TaqMan low density array to simultaneous identification of multiple animal-derived components

Huizhen YU¹(

),Hanli HOU²,Wenxiu YIN¹,Quan ZHANG¹,Chao SUN¹,Mingzhe ZHANG¹,Xiaofeng ZHANG¹,Li MIAO³,Shan WU¹(

)

^1.Zhejiang Academy of Science and Technology for Inspection and Quarantine, Hangzhou 310012，China
^2.Qianjiang Customs, Hangzhou 310007，China
^3.Technology Center of Zhengzhou Customs, Zhengzhou 450003, China

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摘要：

利用TaqMan微流控阵列（TaqMan^? array microfluidic card）技术，将24种（类）动物物种的实时荧光定量聚合酶链式反应（real-time fluorescence quantitative polymerase chain reaction, RT-PCR）的引物探针集合在同一个阵列上，以实现多物种高通量同步检测，并对该方法的重复性、特异性和灵敏度进行验证。用变异系数（coefficient of variation, CV）表示一块阵列板内，同一物种成分检测的重复性。结果显示：66个CV值中，11个低于1%，15个大于2%，其余40个介于1%～2%之间，没有一个物种成分的CV值高于3%。P值显示的是不同阵列板间同一成分检测的重复性，22个物种成分的P值只有1个小于0.05，显示为板间存在差异，其他21种成分的板间差异均未达到显著水平。因此，本研究对多数物种成分的检测，不论板内还是板间都有较好的重复性。特异性检测中未发现TaqMan微流控阵列中出现假阳性的结果，但有2种成分的检测出现了假阴性。TaqMan微流控阵列的检测灵敏度没有高于单重荧光定量PCR，在22个检测样品中，10个物种成分检测的灵敏度降为原来的10%，1个甚至降低为原来的1%，其余11个灵敏度和单重荧光保持一致。总体而言，TaqMan微流控阵列技术在重复性、特异性和灵敏度方面都能满足物种成分真伪鉴定的需求，为同步鉴定多种动物源性成分提供了一种新的方法。

关键词： TaqMan微流控阵列; 动物源性成分; 同步检测

Abstract:

Taking advantage of TaqMan^? array microfluidic card technology, 24 primer-and-probe sets of real-time fluorescence quantitative polymerase chain reaction (RT-PCR) for identification of 24 different animal species (categories) were assembled in one array to achieve simultaneous detection of multiple animal-derived components. The repeatability, specificity and sensitivity of the method were verified. Coefficient of variation (CV) value was used to indicate the repeatability in one array card when detecting the same component (species). Among 66 CV values, 11 CV values were less than 1%; 15 CV values were more than 2%; and the remaining 40 CV values were between 1% and 2%; but none of them was higher than 3%. P value showed the repeatability among different array cards when detecting the same component. Among the 22 P values of the 22 animal-derived components, only one was less than 0.05, which indicated the significant difference of the CV values of the components between the cards. But none of the resting 21 components showed significant inter-plate differences. Therefore, the vast majority of the components tested in this study showed good repeatability both in-card and inter-card. No false positive results were found in TaqMan array tests, but false negative results were appeared in two component detection. The sensitivity of TaqMan array assay was not higher than that of the individual RT-PCR assay. Ten of the 22 components decreased to 10% of the originals in detection sensitivities; another one component even decreased to 1%. And the remaining 11 components were consistent with that of the individual RT-PCR. However, in general, TaqMan array approach can meet the needs of authenticity identification in terms of repeatability, specificity and sensitivity, which offers promise for rapid and simultaneous identification of multiple animal-derived components.

Key words: TaqMan low density array animal-derived components simultaneous identification

收稿日期: 2019-04-22 出版日期: 2020-05-22

CLC:

TS 207.7

基金资助: 国家重点研发计划(2017YFF0210304);浙江省重点研发计划(2018C02041);国家质量监督检验检疫总局科研项目(2015IK299);河南省科技攻关项目(172102110202)

通讯作者: 吴姗 E-mail: yuhuizhen614@163.com;wss_tea@hotmail.com

作者简介: 虞惠贞（https://orcid.org/0000-0003-1275-2739），Tel：+86-571-81100350，E-mail：yuhuizhen614@163.com

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引用本文:

虞惠贞,侯寒黎,尹文秀,张荃,孙超,张明哲,张晓峰,苗丽,吴姗. 利用TaqMan微流控阵列同步检测多种动物源性成分[J]. 浙江大学学报（农业与生命科学版）, 2020, 46(2): 189-200.

Huizhen YU,Hanli HOU,Wenxiu YIN,Quan ZHANG,Chao SUN,Mingzhe ZHANG,Xiaofeng ZHANG,Li MIAO,Shan WU. Application of TaqMan low density array to simultaneous identification of multiple animal-derived components. Journal of Zhejiang University (Agriculture and Life Sciences), 2020, 46(2): 189-200.

链接本文:

http://www.zjujournals.com/agr/CN/10.3785/j.issn.1008-9209.2019.04.221 或 http://www.zjujournals.com/agr/CN/Y2020/V46/I2/189

中文名 Chinese name	学名 Scientific name	英文名 English name	样品部位 Part of sample	采集地 Collecting location
黄牛^*	Bos taurus	Yellow cattle	肌肉	市场上购买
水牛^*	Bubalus ubalis	Water buffalo	肌肉	市场上购买
家畜骆驼^*	Camelus ferus bactrianus	Domestic camel	肌肉	市场上购买
绵羊^*	Ovis aries	Sheep	肌肉	市场上购买
家猪^*	Sus scrofa domesticus	Pig	肌肉	市场上购买
马^*	Equus ferus	Horse	肌肉	市场上购买
驴^*	Equus asinus	Donkey	肌肉	市场上购买
中华田园犬^*	Canis lupus familiaris	Dog	肌肉	市场上购买
家兔^*	Oryctolagus cuniculus f. domesticus	Rabbit	肌肉	市场上购买
赤狐^*	Vulpes vulpes	Red fox	肌肉	本实验室保存^**
梅花鹿	Cervus nippon	Sika deer	骨骼	本实验室保存^**
牦牛	Bos grunniens	Yak	肌肉	市场上购买
鸡^*	Gallus gallus domesticus	Chicken	肌肉	市场上购买
鸭^*	Anas	Duck	肌肉	市场上购买
鹅^*	Anser cygnoides orientalis	Goose	肌肉	市场上购买
家鸽^*	Columba livia domesticus	Pigeon	肌肉	市场上购买
火鸡^*	Meleagris gallopavo	Turkey	肌肉	市场上购买
日本鹌鹑^*	Coturnix japonica	Quail	肌肉	市场上购买
中华鹧鸪^*	Francolinus pintadeanus	Chinese francolin	肌肉	市场上购买
麻雀	Passer montanus	Eurasian tree sparrow	内脏	本实验室保存^**
爪哇金丝燕	Aerodramus fuciphagus	Thunberg’s swiftlet	唾液	市场上购买
东星斑^*	Plectropomus leopardus	Leopard coral grouper	肌肉	本实验室保存^**
大西洋鲑^*	Salmo salar	Atlantic salmon	肌肉	市场上购买
黄鳍金枪鱼^*	Thunnus albacores	Yellow fin tuna	肌肉	市场上购买
大西洋鳕^*	Gadus morhua	Atlantic cod	肌肉	市场上购买
异鳞蛇鲭^*	Lepidocybium flavobrunneum	Oil fish	肌肉	市场上购买
细鳞壮鳕	Albatrossia pectoralis	Giant grenadier	肌肉	市场上购买
马舌鲽	Reinhardtius hippoglossoides	Greenland halibut	肌肉	市场上购买
水稻	Oryza sativa L.	Rice	可食用部分	市场上购买
白菜	Brassica rapa pekinensis	Cabbage	可食用部分	市场上购买
单增李斯特菌	Listeria monocytogenes			本实验室保存^**
创伤弧菌	Vibrio vulnificus			本实验室保存^**
粪肠球菌	Enterococcus faecalis			本实验室保存^**
金黄色葡萄球菌	Staphylococcus aureus			本实验室保存^**

表1 用于检测的样品信息

图1 TaqMan低密度阵列板的设计

目标物种^* Target species^*	引物和探针序列（5′→3′） Sequences for primers and probes (5′→3′)	目的基因 Target gene	文献 Reference
黄牛 Bos taurus	F: AACCAAAGGAGGATTTAGCAGT R: ACGACTTGTCTCCTCTCATGT P: FAM-AATTAGGCCATGAAGCACGC-BHQ1	线粒体12S rRNA基因	本研究
水牛 Water buffalo	F: TTCATTGAYCTCCCTGCTCC R: GGAATAGGCCGGTGAGGATT P: FAM-ACTTTGGCTCTCTCC-BHQ1	线粒体ATPase 8基因	[8]
骆驼 Camelus	F: GACTAATATTCGAAAATCCC R: CTCCTAGGAGGGAGCCGAAGTTTCAC P: FAM-CAACGCATTCATTGACCTTCCAGCTCCAT-BHQ1	线粒体Cyt b基因	[9]
羊 Sheep and goat	F: ACACAACTTCTACCACAACCC R: AAACAATGAGGGTAACGAGGG P: FAM-ACACCGAAACAAAATACTCCTTGAGAAACA-BHQ1	线粒体Cyt b基因	[10]
猪 Pig	F: TTTGTGCATGACTGCGTCAAC R: CTTGGTGGTCGTGGTCACTGT P: FAM-CACCGTCAAGCAGC-BHQ1	朊蛋白基因	[10]
驴 Donkey	F: CCTCAGCACTCCCCCTCAT R: AAGGATAAGGGCTAATACACCA P: FAM-CCAGAATGGTATTTCCTATTTGCTTACGCC-BHQ1	线粒体Cyt b基因	[11]
马 Horse	F: TCTAGCCCTAGCCGTGCGGCTAACC R: TAGGATGATAAACGTAATAAGGGCTG P: FAM-CGCCGGACACCTCCTAATACACCTC-BHQ1	线粒体ATPase 6基因	[12]
狗 Canis	F: TCCAGGTAAACCCTTCTTCCC R: TTACGAGCAAGGGTTGATGGT P: FAM-ATCACTTAGTCCAATAAGG-BHQ1	线粒体D-loop基因	[13]
兔 Rabbit	F: TAATCGTCACCGCACATGCC R: CTATGTCAGGAGCCCCAATTATCA P: FAM-ACAAGCCAGTTCCCGAAGCCTCCA-BHQ1	线粒体COI基因	[14]
狐 Fox	F: TGGAGCATCAGTAGACCTTACAATTT R: GGCGGGAGGTTTTATATTGATAATAG P: FAM-CCCTGCACCTGGCCGGAGTC-BHQ1	线粒体COI基因	[15]
鸡 Chicken	F: CCCTCCTCCTTTCATCCTCAT R: GTCATAGCGGAACCGTGGATA P: FAM-CTATGAATCCGGGCCTC-BHQ1	—	[16]
鸭 Anas platyrhynchos and Anas poecilorhyncha	F: CCTAGCCTCAGGCGTAACTG R: CAGACGGTTAGGAAGGTGGA P: FAM-CATGAAGCCCCATTCTCAAT-BHQ1	线粒体ATPase 6基因	本研究
鹅 Goose	F: CCACCGCGAATCCTCAAT R: TCTTGTTTTTGGAGGATTTGTTCTC P: FAM-CCATCGCAGAGCCTGGTCGATATTTTT-BHQ1	—	[16]
鸽 Columba livia	F: CACGTGAAACCAGCAACTCG R: ATGGTGAGGTATGACCCCGA P: FAM-GGTTCCTATGTCAGGGCCAT-BHQ1	线粒体D-loop基因	本研究
火鸡 Meleagris gallopavo	F: TGATGAAACTTCGGCTCCCT R: CGCGTCCAATGTGTAGGAAG P: FAM-CGGCCTCCTACTAGCCATAC-BHQ1	线粒体Cyt b基因	本研究
鹌鹑 Quail	F: GGATCACTTTGCCCACTTTAA R: GGGTGACGGGCGGTATG P: FAM-ATGGCCCTGAGGCAC-BHQ1	—	[16]
鹧鸪 Chinese francolin	F: CAGGCTTTACCCTACACCCC R: AGTATCGGCGAGGTATGCCG P: FAM-AGCACACTTCGGAGTTATGTTTGT-BHQ1	线粒体COI基因	[17]
石斑鱼 Grouper	F: TGGTGGCTTTGGAAACTGACT R: ACCCCAGAAGAAGCGAGAAGGA P: FAM-AGCTTCTGACTTCTTCCTCCATCCTTCCTG-BHQ1	线粒体COI基因	[18]
大西洋鲑 Salmo salar	F: CCCACCACCGACAAATAACG R: CTTTCCCTGTGAGCACTCCT P: FAM-GGGAACTGTAGCCCAGAACA-BHQ1	线粒体12S rRNA基因	本研究
金枪鱼 Tuna fish	F: CCTTACAGGACTATTCCTCGC R: CCGATGTTTCATGTTTCTTTG P: FAM-TCGAGACGTTAACTACGGCTG-BHQ1	线粒体Cyt b基因	[19]
鳕鱼 Cod fish	F: GGGAAGAGCTTTGAGTAGAGGTGA R: AAGGGCTGAACTCCTATTCATTTC P: FAM-CGAGCCCAGTTATAGCTGGTTGCCTG-BHQ1	线粒体16S rRNA基因	[20]
异鳞蛇鲭 Lepidocybium flavobrunneum	F: GGTCGAGGTGTAGCGTATGA R: TAGGTTGCTTGGGGAGAGTG P: FAM-CAGCGAATACGAACGATGCA-BHQ1	线粒体12S rRNA基因	本研究
哺乳动物 Mammal	F: CCTCAGCAGGGTCTTCACCA R: TTCAGCTTCTCGTAGACNCG P: FAM-CAGCCTGGTGTTGGCACCTCGGA-BHQ1	生长激素基因	[9]
真核生物 Eukaryotes	F: CCTGAGAAACGGCTACCAT R: CGTGTCAGGATTGGGTAAT P: FAM-TGCGCGCCTGCTGCCTTCCT-BHQ1	核糖体18S rRNA基因	[10]

表2 本研究中引物和探针序列

表3 用于TaqMan微流控阵列特异性检测的DNA样品组合

表4 用于TaqMan微流控阵列灵敏度检测的DNA样品组合

表5 TaqMan微流控阵列的重复性检测

表6 TaqMan微流控阵列特异性验证

表7 TaqMan微流控阵列与普通单重荧光定量PCR检测灵敏度比较

1	ONYANGO C O, LOPAREV V, LIDECHI S, et al. Evaluation of a TaqMan array card for detection of central nervous system infections. Journal of Clinical Microbiology, 2017,55(7):2035-2044. DOI:10.1128/JCM.02469-16 doi: 10.1128/JCM.02469-16
2	LIU J, GRATZ J, AMOUR C, et al. A laboratory-developed TaqMan array card for simultaneous detection of 19 enteropathogens. Journal of Clinical Microbiology, 2013,51(2):472-480. DOI:10.1128/JCM.02658-12 doi: 10.1128/JCM.02658-12
3	PHOLWAT S, LIU J, STROUP S, et al. Integrated microfluidic card with TaqMan probes and high-resolution melt analysis to detect tuberculosis drug resistance mutations across 10 genes. mBio, 2015,6(2):e02273-14. DOI:10.1128/mBio.02273-14 doi: 10.1128/mBio.02273-14
4	CRUZEIRO G A V, SALOM?O K B, DE BIAGI JR C A O, et al. A simplified approach using TaqMan low-density array for medulloblastoma subgrouping. Acta Neuropathologica Communications, 2019,7:33. DOI: 10.1186/s40478-019-0681-y doi: 10.1186/s40478-019-0681-y
5	SATIH S, SAVINEL H, RABIAU N, et al. Expression analyses of nuclear receptor genes in breast cancer cell lines exposed to soy phytoestrogens after BRCA2 knockdown by TaqMan low-density array (TLDA). Journal of Molecular Signaling, 2009,4:3. DOI:10.1186/1750-2187-4-3 doi: 10.1186/1750-2187-4-3
6	HUI A B Y, SHI W, BOUTROS P C, et al. Robust global micro-RNA profiling with formalin-fixed paraffin-embedded breast cancer tissues. Laboratory Investigation,2009,89(5):597-606. DOI:10.1038/labinvest.2009.12 doi: 10.1038/labinvest.2009.12
7	ZHANG L, ZHAO W P, VALDEZ J M, et al. Low-density TaqMan miRNA array reveals miRNAs differentially expressed in prostatic stem cells and luminal cells. The Prostate, 2010,70(3):297-304. DOI:10.1002/pros.21064 doi: 10.1002/pros.21064
8	国家认证认可监督管理委员会.食品及饲料中常见畜类品种的鉴定方法第7部分:水牛成分检测实时荧光PCR法:SN/T 3730.7—2013.北京:中国标准出版社,2014. Certification and Accreditation Administration of the People’s Republic of China. Identification of Domestic Animal Ingredient in Food and Feed-Part 7: Detection of Buffalo Ingredient-Real-Time PCR Method: SN/T 3730.7—2013. Beijing: Standards Press of China, 2014. (in Chinese)
9	国家认证认可监督管理委员会.出口食品中骆驼源性成分的检测实时荧光PCR法:SN/T 4418—2016.北京:中国标准出版社,2017. Certification and Accreditation Administration of the People’s Republic of China. Detection of Camelus Ingredient in Export Food-Real-Time PCR Method: SN/T 4418—2016. Beijing: Standards Press of China, 2017. (in Chinese)
10	全国畜牧业标准化技术委员会.明胶中牛、羊、猪源性成分的定性检测方法实时荧光PCR法:GB/T 25165—2010.北京:中国标准出版社,2011. National Animal Husbandry Standardization Technical Committee. Protocol of Identification of Bovine, Caprine, Ovine and Porcine Derived Materials in Gelatin-Real-Time PCR Method: GB/T 25165—2010. Beijing: Standards Press of China, 2011. (in Chinese)
11	中华人民共和国商务部.肉及肉制品中动物源性成分的测定实时荧光PCR法:SB/T 10923—2012.北京:中国标准出版社,2013. Ministry of Commerce of the People’s Republic of China. Species Identification of Animal Derived Materials in Meat and Meat Products-Real-Time PCR Method: SB/T 10923—2012. Beijing: Standards Press of China, 2013. (in Chinese)
12	国家认证认可监督管理委员会.食品及饲料中常见畜类品种的鉴定方法第5部分:马成分检测实时荧光PCR法:SN/T 3730.5—2013.北京:中国标准出版社,2014. Certification and Accreditation Administration of the People’s Republic of China. Identification of Domestic Animal Ingredient in Food and Feed-Part 5: Detection of Horse Ingredient-Real-Time PCR Method: SN/T 3730.5—2013. Beijing: Standards Press of China, 2014. (in Chinese)
13	国家认证认可监督管理委员会.食品及饲料中常见畜类品种的鉴定方法第2部分:狗成分检测实时荧光PCR法:SN/T 3730.2—2013.北京:中国标准出版社,2014. Certification and Accreditation Administration of the People’s Republic of China. Identification of Domestic Animal Ingredient in Food and Feed-Part 2: Detection of Canis Ingredient-Real-Time PCR Method: SN/T 3730.2—2013. Beijing: Standards Press of China, 2014. (in Chinese)
14	全国饲料工业标准化技术委员会.动物源性饲料中兔源性成分定性检测方法实时荧光PCR方法:GB/T 21102—2007.北京:中国标准出版社,2007. National Feed Industry Standardization Technical Committees. Identification of Rabbit Derived Materials in Animal-Originated Feedstuffs-Real-Time PCR Method: GB/T 21102—2007. Beijing: Standards Press of China, 2007. (in Chinese)
15	国家认证认可监督管理委员会.食品及饲料中常见畜类品种的鉴定方法第3部分:狐狸成分检测实时荧光PCR法:SN/T 3730.3—2013.北京:中国标准出版社,2014. Certification and Accreditation Administration of the People’s Republic of China. Identification of Domestic Animal Ingredient in Food and Feed-Part 3: Detection of Vulpes Ingredient-Real-Time PCR Method: SN/T 3730.3—2013. Beijing: Standards Press of China, 2014. (in Chinese)
16	国家认证认可监督管理委员会.饲料中禽源性成分检测方法实时荧光PCR方法:SN/T 2727—2010.北京:中国标准出版社,2011. Certification and Accreditation Administration of the People’s Republic of China. Determination of Poultry Derived Materials Feedstuff-Real-Time Fluorescent PCR Method:SN/T 2727—2010. Beijing: Standards Press of China, 2011. (in Chinese)
17	国家认证认可监督管理委员会.食品及饲料中常见禽类品种的鉴定方法第6部分:鹧鸪成分检测实时荧光PCR法:SN/T 3731.6—2013.北京:中国标准出版社,2014. Certification and Accreditation Administration of the People’s Republic of China. Identification of Poultry Ingredient in Food and Feed-Part 6: Detection of Alectoris Ingredient-Real-Time PCR Method: SN/T 3731.6—2013. Beijing: Standards Press of China, 2014. (in Chinese)
18	国家认证认可监督管理委员会.出口食品中常见鱼类及其制品的鉴伪方法第1部分：石斑鱼成分检测实时荧光PCR法:SN/T 3589.1—2013.北京:中国标准出版社,2014. Certification and Accreditation Administration of the People’s Republic of China. Identification of Fish Species in Export Food-Part 1: Detection of Grouper Ingredient-Real-Time PCR Method: SN/T 3589.1—2013. Beijing: Standards Press of China, 2014. (in Chinese)
19	国家认证认可监督管理委员会.出口食品中常见鱼类及其制品的鉴伪方法第6部分：金枪鱼成分检测实时荧光PCR法:SN/T 3589.6—2013.北京:中国标准出版社,2014. Certification and Accreditation Administration of the People’s Republic of China. Identification of Fish Species in Export Food-Part 6: Detection of Tuna Fish Ingredient-Real-Time PCR Method: SN/T 3589.6—2013. Beijing: Standards Press of China, 2014. (in Chinese)
20	国家认证认可监督管理委员会.出口食品中常见鱼类及其制品的鉴伪方法第7部分：鳕鱼成分检测实时荧光PCR法:SN/T 3589.7—2013.北京:中国标准出版社,2014. Certification and Accreditation Administration of the People’s Republic of China. Identification of Fish Species in Export Food-Part 7: Detection of Cod Fish Ingredient-Real-Time PCR Method: SN/T 3589.7—2013. Beijing: Standards Press of China, 2014. (in Chinese)

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