CN109337991A - 一种基因Gcg在检测家鸡源性成分中的应用及其试剂盒 - Google Patents
一种基因Gcg在检测家鸡源性成分中的应用及其试剂盒 Download PDFInfo
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Abstract
本发明公开一种基因Gcg在检测家鸡源性成分中的应用及其试剂盒，筛选出一个家鸡通用基因Gcg，其核苷酸序列如SEQ ID NO.1所示，其位于家鸡基因组染色体上，单拷贝易定量，在家鸡基因组中拷贝数恒定，不存在等位基因变异，以该基因为靶序列，引入特异性引物和探针作为试剂盒，可快速、灵敏地检测食品中的家鸡源性成分，灵敏度高，特异性强，操作简单、成本低廉，反应结果易于观察，非常适用于现场实时检测，在动物源性食品监督与精准检测肉制品掺假方面应用前景广阔。
Description
技术领域
本发明涉及生物物种鉴定和PCR检测技术领域，具体的说涉及一种基因Gcg在检测家鸡源性成分中的应用及其试剂盒。
背景技术
中国是肉类消费大国，随着社会的发展、居民生活水平的提高，人们的饮食结构也在逐渐发生变化，动物性蛋白质在饮食中所占的比例呈现逐渐升高的趋势。经济和产品技术加工的发展，很多食品被非法添加各种化学原料、添加剂、激素等，甚至很多商家为谋求高额利益进行食品掺假行为或直接以次充好，特别是以价格低廉的马肉、猪肉、鸡肉或其他动物肉类冒充价格高昂的牛肉、羊肉，赚取高额利润。这严重危害了消费者的利益和身体健康，因此对食品种类的准确鉴定至关重要。
家鸡是重要的农业动物，为人们提供大量蛋、肉等优质食品，而且在生理、发育、衰老和疾病等研究领域也是重要的模式动物，与人们生活也有着密切的联系。家鸡是人类驯化较早的家禽，而我国是最早驯化家鸡的国家之一。在长期的生产实践中，已经选育出众多适应于不同生境条件和饲养条件的家鸡品种。
然而越来越多的商家为获取高额利益，用激素和抗体喂养禽类，使其加速增长，饲养过程中在禽饲料中添加禽成分存在传播禽流感的风险，影响畜禽健康生产，威胁畜牧业安全。更有不法商家用便宜的肉制品甚至蛋白制品掺假家鸡肉，以谋取暴力等。而家鸡肉污染可能引发严重食品安全问题，利用家鸡的内标准基因和分子技术检测家鸡肉掺假是一种有效的检测手段。
目前，内标准基因已被广泛地用于鉴别食品掺假，但如何筛选出合适的内标准基因显得尤为重要。目前国内外的肉制品掺假检测技术多是针对线粒体上的基因设计特异引物来进行实时荧光定量PCR扩增，由于线粒体基因为多拷贝基因，其检测灵敏度高，但同时在区分由于销售、运输等过程所产生的无意识交叉污染与有意识的非法添加时存在困扰。另外，高拷贝数线粒体DNA的浓度无法和基因组DNA的浓度相对应，因此无法对样品进行精确定量分析，同时由于线粒体基因同源性极高，难以通过普通PCR实现定性检测。因此，该方法只能借助荧光定量PCR实现肉类掺假的筛选鉴别。若要鉴别低浓度的无意交叉污染和有意非法添加并明确掺假比例实现快速筛查，则要选择染色体上的低拷贝基因作为肉类内标准基因。
发明内容
本发明的目的是提供一种基因Gcg在检测家鸡源性成分中的应用，以基因Gcg作为内标准基因，该内标准基因Gcg在食品或饲料的家鸡成分鉴定中的应用，基因Gcg的核苷酸序列如SEQ ID NO.1所示。
本发明还提供一种用于检测家鸡源性成分的试剂盒，以基因Gcg为靶基因建立的试剂盒，包括基因Gcg的特异性引物，采用PCR方法检测内标准基因Gcg的特异性引物，特异性引物的核苷酸序列如SEQ ID NO.2和SEQ ID NO.3所示。
所述试剂盒还包括探针，探针的核苷酸序列如SEQ ID NO.4所示。
本发明是以Gcg基因作为内标准基因在检测家鸡源性成分中的应用，首次在染色体上筛选出家鸡源性内标准基因，且拷贝数为单拷贝，内标准基因的选择一般要求拷贝数低且稳定，一般动物内标准基因常选择在线粒体上，这样的内参基因拷贝数多，不易于定量，本发明选择染色体上的基因做为内标准基因，其拷贝数低易于定量，相比于线粒体上的基因，突变率更低。
本发明采取southern杂交的方式，进行拷贝数的鉴定，Sma 1和Nhe 1酶切的DNA片段中均出现了一条条带，进而确定了目标Gcg基因的拷贝数为单拷贝。
本发明确定用于检测食品及饲料中家鸡源成分的基因Gcg，设计了用于检测该基因的定性PCR和定量PCR的引物和/或探针作为试剂盒，建立了检测家鸡源性成分的相应PCR方法，该方法费时少，特异性好，灵敏度高，操作简单，不需要专业人员操作，结果易于观察，非常适于基层食品监督检验使用。
附图说明
图1为基因同源性分析；
图2为家鸡的Southern杂交结果（图中1-Sma 1酶切普通家鸡；2-Sma 1酶切乌鸡；3-Nhe1酶切普通家鸡；4-Nhe 1酶切乌鸡）；
图3为Gcg基因的定性PCR验证特异性（泳道1-23，图中: 1-驴；2-鸭；3-火鸡；4-野鸡；5-猪；6-牛；7-羊；8-马；9-普通家鸡；10-乌鸡；11-鹅；12-狗；13-兔；14-鼠；15-鱼；16-牦牛；17-水牛；18-骆驼；19-貂；20-鹿；21-人；22-猴；23-阴性；M-DNA Marker DL 2000）；
图4为Gcg基因的定量PCR验证特异性（其中1是普通家鸡；2是乌鸡）；
图5为Gcg基因的荧光定量PCR扩增曲线（其中1为100ng/μL，2为10ng/μL，3为1ng/μL，4为0.1ng/μL，5为0.01ng/μL，6为阴性，每个浓度做三个平行）；
图6为Gcg基因的荧光定量PCR标准曲线。
具体实施方式
下面结合具体实施例进一步说明本发明的内容，但不应理解为对本发明的限制，在不背离本发明精神和实质的情况下，对本发明方法、步骤或条件所作的修改或替换，均属于本发明的范围。若未特别指明，实施例中所用的技术手段为本领域技术人员所熟知的常规手段。
猪(Sus scrofa)，牛(Bos taurus)，羊(Ovis aries)，普通家鸡(Gallus gallus)，野鸡（Phasianuscolchicus），火鸡（Meleagris gallopavo），乌鸡(Gallus domesticus brisson)，鸭(Anas platyrhynchos)，鹅(Goose calicivirus),狗(Canis lupus familiaris)，兔(Oryctolagus cuniculus)，牦牛(Bos mutus)，黄鱼（Pseudosciaena polyactis）为超市购买；马(Equus caballus)，驴(Equus asinus)为北京农贸市场购买；老鼠(Mus musculus)由中国农业大学食品安全与分子生物学实验室提供；水牛(Bubalus bubalis)，貂(Martes zibellina)，骆驼(Camelus ferus)，鹿(Cervus)由天津出入境检验局的李宗梦博士提供；猴(Macaca fascicularis)由中国农业科学院动物所提供；人(Homo sapiens)胎盘组织由空军总医院提供。
实施例1 家鸡源通用内标准基因Gcg的筛选
通过搜索GenBank中关于家鸡的基因信息，对基因组信息进行BLASTn和DNAman软件同源性、特异性分析，筛选出Gcg基因，该基因位于家鸡基因组染色体7号上，由图1所示，经BLASTn序列同源性分析，此Gcg基因片段在数据库中基因同源性最低；将家鸡的Gcg基因片段与非家鸡物种DNA序列进行多序列特异性分析，具有较高的家鸡源特异性；最后通过对序列进行整合，确定最终的特异性目标基因Glucagon（Gcg）基因可以作为内标准基因；该Gcg基因片段的核苷酸序列如SEQ ID NO.1所示。
实施例2 家鸡源性成分通用内标准基因的验证
1、针对实施例1筛选的内部基因Gcg，通过Primer Express 3.0设计软件（美国生命技术公司）针对Gcg基因设计PCR及定量PCR引物和探针：
Gcg-F: CAAAAAAAAAAAACAAAAAACAAAAACCTACAGA，SEQ ID No. 2；
Gcg-R: TCTGTAATGATAGATTTGTATAAAATGGGACTG，SEQ ID No. 3；
Gcg-P: ACAACACTAACAAAAGGAAAATGGCCAGGC，SEQ ID No. 4，其5' 端标记FAM荧光基团和3' 端标记TAMRA荧光基团。
定性PCR检测，使用Gcg-F/R引物对，其PCR扩增产物目的片段长度为196 bp；定量PCR检测，使用Gcg-F/R引物对结合Gcg-P荧光探针；Southern杂交，使用Gcg-F/R引物对，用扩增产物作为杂交探针进行拷贝数检测。
、家鸡Gcg基因的拷贝数分析
采取southern杂交的方法，对筛选的Gcg基因在家鸡基因组DNA中的拷贝数进行鉴定，结合Ensembl基因信息数据库数据，确定了Gcg基因的拷贝数为单拷贝。
理想的内标准基因在相应的物种中应该拷贝数恒定并不存在等位基因变异。为了鉴定Gcg基因在家鸡中的拷贝数，本发明使用引物Gcg-F/R，即SEQ ID No. 2和SEQ ID No.3，扩增一段196 bp DNA片段，该片段作为探针用于Southern blot，用Sma 1和Nhe 1分别切割家鸡的基因组DNA，然后和198 bp DNA片段杂交，由图2可知Sma 1和Nhe 1酶切的DNA片段中均出现一条条带，所以该片段在家鸡中为单拷贝。
、家鸡Gcg基因的特异性鉴定及其等位基因变异的分析
利用设计的引物对Gcg-F/R对2种家鸡物种（普通家鸡、乌鸡）和20种非家鸡物种（猪、牛、火鸡、羊、马、驴、鸭、鹅、狗、野鸡、兔、鼠、鱼、牦牛、水牛、骆驼、貂、鹿、猴、人）进行定性PCR扩增，定性PCR反应体系见表1所示，用2%的琼脂糖凝胶对扩增产物进行电泳分析，PCR扩增反应程序：95ºC预变性5min；95ºC预变性30s，60ºC预变性45s，72ºC预变性1min，35个循环；72ºC延伸10min；同时进行定量PCR分析，定量PCR反应体系见表2所示，反应程序：95ºC预变性10min；95ºC预变性15s，60ºC预变性1min，40个循环；60ºC采集信号。
结果表明，只有家鸡物种（普通家鸡和乌鸡）均能扩增出196 bp的片段，符合所设计引物对应的片段大小，而其它20种非家鸡物种均无特异性扩增条带，如图3所示；用引物Gcg-F/R和探针Gcg-P进行定量PCR对该22种物种进行检测，只有两个家鸡物种能检测到信号，而20种非家鸡物种没有检测到信号，如图4所示，这表明该基因以及对应的引物可以用来特异性地检测家鸡及家鸡源性成分。
表1 定性PCR反应体系
表2 定量PCR反应体系
4、定量PCR灵敏度验证
为了评定荧光定量PCR的灵敏性，将家鸡的基因组DNA浓度10倍稀释7个梯度，分别为50ng/μL，5ng/μL，0.5ng/μL，0.05ng/μL，5pg/μL，0.5pg/μL，0.05pg/μL，进行荧光定量PCR，扩增曲线如图5，取5个点绘制标准曲线，得到的标准曲线如图6所示，当DNA模板浓度最低至10pg时，能检测到荧光信号，因此，该荧光定量体系的检测限是10pg DNA，也就是8个拷贝数，同时给出了关于Cq值与基因组浓度的对数之间线性方程，线性方程为：y = -3.196x +32.481，R2 =1，y代表Cq值，x代表基因组浓度的对数值，R2代表线性相关系数，根据R2值可知家鸡基因组DNA的拷贝数和Ct值间具有良好的线性关系，由前面拷贝数鉴定可Gcg基因在家鸡均为单拷贝，由此可推测，该基因和定量 PCR体系也适合用于家鸡的定量检测，其检测限为8个拷贝数。
、鸡肉加工品检测
为验证内标准基因Gcg以及建立的定量PCR检测技术在实际样品检测过程中的准确性，对市售12种鸡肉加工制品进行检测，鸡肉加工品检测结果显示只有家鸡肉加工品能收集到荧光信号，如表3所示，野鸡肉、火鸡肉三明治在核酸提取完全的情况下没收集到荧光信号，表明基于Gcg基因建立的家鸡定量检测体系在种间区分方面具有特异性，同时，张君雅小妹妹炭烧鸡汁点心面和墨西哥辣鸡点心面2种方便面食品也没收集到荧光信号，该两种食品配方均显示不包含鸡源性成分，结果与实际相符，因此，基于Gcg基因建立的鸡定量检测体系在实际样品含有鸡源性成分的情况下，均能检测出，进一步证明了该方法的灵敏性高，可以用于鸡肉加工品的检测。
表3 鸡肉加工品检测结果
序列表
<110> 昆明理工大学
<120> 一种基因Gcg在检测家鸡源性成分中的应用及其试剂盒
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 12876
<212> DNA
<213> 家鸡(Gcg)
<400> 1
tccagaccct tcttcctcct gagcagaact gaaactgaaa gtgaagtcct taatatttaa 60
tatctctgct tgatgcacaa tattctatat atataataat catattttat ttctagtgca 120
cttttcattt ttaaaagaat tatcatatgt caaaaaatcc ataagggaag ctctgtagga 180
attccttgac agaggatatc tcagttgttg cttcactgac ccgtattcct tactggtggg 240
ctttttttca gctgtcagga acactgacaa gatttgtcat tttctcctac tgttgtcaca 300
agatggctaa acacagttta tgtcactgct gccccatcac aggcatgaat tttcccccca 360
tctattagtg aatagtttag gactttggca atatgtgtac agaacataca caggtatgaa 420
actcacacct atatttttgt tgtaattgtt tccttgtagg gaacagagat gtgagcaaaa 480
ggcttatctt gtataaactt gtagaccatg gtgtgaaacc caatttacaa tggaagtctg 540
gcatacagat ctccagtgag tatgagttat cattcagtag agttggcgta atgcatctaa 600
gaataatcaa gtcttttgtg ttgttaaatt tgacctgagg taggctttgc atagtgagta 660
gctattgctg tagtgcttca aagtagaagt gcttctttat gttcgtaata ttggcataag 720
gctacctgaa aaattaacca agcactttgg gggggtagtg ctggttattg ttaatagtgt 780
attaaattta taaatttaga cttgaaattg ataggttttg ttgatgctac taatgtcgac 840
aggttcagca ttttacttaa tactaagtgc tttgcttaat actaatgtga caattcagaa 900
tttggagaaa ctgctgcctt aatcctaagc acctctagcc aactgtttat catcctaatg 960
cccttctgtt tgatatttac ttctttgaac attgctacct tactcttttc gtaaatccat 1020
gttagcattg atgtcataaa gtaagcagaa tgaatgtgtg aaattgcttg taaaattatc 1080
aaatgaaatg aatagcaaat acgatgactt tttttttaaa gttggataag gaaactgtca 1140
gcaatgatgc actctggtaa tgcttactag agctgaacag tatatatata agtctgtgca 1200
aaagaggcag ctaggcagat ctcaggatcc tcaaggcttt ccatagttcc agaagagaga 1260
gtagcagatc tttctccaac tgctacatat cacttcaaaa tataaaaggt aagatttatg 1320
tgaagtagta gtacaattca tctgagaaag ctgaatgcat tttctgaatt gtattttcca 1380
aagattgcaa cagagatgca ggatctttca acaaattgtt gttttcttct gtggttatac 1440
tccttaatgt ttggaattaa ttacatcacc atttaatttt ttttaatcta ttgagttaat 1500
ttaacagaat aatcccaaaa tggaacatca actactagga ttgcagaagc aattgaaaga 1560
aggaaggcat gtaaagatga aaaacaggca ttgtagtaag aatttaaatg tgaaatacaa 1620
aactattgat acttatgttt ttatctactt cagggatatt taatgaatgt tatttcaaaa 1680
atattcattc cttacagctg cttcctaaat atgaatcact atagattaac ttgggaaaat 1740
gcaaacaaaa taaatggctc catatggaca ctgactaaaa actgtccaaa acagcttgtc 1800
accccatcac cctacaggtg accactccgt tgccttactt tccaattagt ccacctcaaa 1860
aaaactgtta ttttcatatc actgtaagat ctcagtggga aattttatag cattaaaaaa 1920
aaaaaaacaa aaaaaacctt tcaatggtta ttgtgtttat tatataaaat ttaatttagt 1980
tttctggtct ggactttctt aggccagaat gattgattgt ttttttatag aactctccac 2040
attgctgcag ataaatagac acaaagcaca tctatccaaa acagtaatac actgataact 2100
agaaataatt gttatctgca attgttgctt cagaaggaat ctactttatt aattcttaaa 2160
ttttcatata taatattcct tttttttact gatgtcgttc catgcttttt atttttaaac 2220
tggaaaagga tacaaatctg acttttgaaa gaattttttt aaaaaactca ataacattaa 2280
gttagtttaa ctcgcacaca gtgtgcaaaa gataactaca gcaaaatata tgggaaaacc 2340
agaaaaatca gattgataag aaaataatat tcatttcata ttttctcaca gctcaaagtg 2400
agatcttttt gattagaaat gtttttcata agcactttct aagtgaagtt agctttagtt 2460
ttcctgcacc ctaacagtag aatccctgtc tttttctttt tataatagaa atttgttctt 2520
taaatactat aatttctttt acttgtagaa aactgctgat gctggtttgg gttagaaatg 2580
acaagtgcta aactctaaac aaactaagca gattctaaac ttttaggaaa atatgtttga 2640
cagaggttta acagccagtc agtacatttt aaaatgttta ttctattgat ggccaggttt 2700
cttttacttg aaaatgtgat taatttccaa aactgcattt atacatcttt ctttttatta 2760
atctcctcag aatttgattc ttaaaaattt agaaagaagt tcaatgtaca tttaaattta 2820
aacatgtttt taatgcataa agtagctagt gggactgttc atatctgaaa gctctatagg 2880
actgagaccc aggagttagc tcaagatatt attaaatcac tgaaaatctg tttgtcagtt 2940
acaatgggct ttaaatattt attttcaagt tttatgcatc agaatagctt ttttgtctga 3000
tactattata aacagattag attaatgatg aggctatttc tttcactcca tttcagtgac 3060
ttaggtttat gtctaaaccg tatcatgatt taacagtggt acaggcagta gtaaagtata 3120
gatcctgttc tagtttttca acttaagtaa atgtatacta gtttgtttgt ataccatcac 3180
acatgagaaa attacttacg tatagtgcaa taccaggagt ctatttggtg tctgggctaa 3240
ctcatttata gctatgtcag ctagcatgag ggtcactaca cagaactcaa gccagttttg 3300
tgatcatagc aatctcccta agaataaggc agcgcctttg ttgtgtcact gaaagcctat 3360
gaaatgtgaa attatgcatt ctttaggact aacattggat gtcagtgttg caccactaaa 3420
ttacagctcc aagtgaaata gcaaaaagag agtttgatat tatagttaag attataacag 3480
gactgaatga ttacatagcc actgcattat ttgttgttac tacaggtatg ttttatggag 3540
atgtaggaat caaatctcat atatgagagc attacaaaag gcagaaaaca cattatacac 3600
agtgaaaaaa aaaaaaaaaa aaaaaaagaa cagtactgtc agtcgctccc ggtagagaca 3660
aggaaattga ctccattgtt tgatataata aaatccacca agaagtctta tatttttcat 3720
tttcccattt tttaataaga ttttgaatga ctaaatattt tgatattttg atattttgag 3780
gtattgaagc aaaatagcat tctatggatt cctccaggcc attgtatatt taaatccagc 3840
ctattatgac aaggcagaca gagaatatat atgttttctt tcaaattgct ttatttcata 3900
aattgaattt atatatcaaa ttattggaga agtttagtgc agattacata tagtgtttgg 3960
tgtttatagc tgggtcctat tataccacct atgactttac ttcagtgcat tcatctttcc 4020
tatttcactt tgtttttcag actttaacat aaaattctat aggaaaattc catggatggc 4080
agaagggctc catcaaatag tcaagtatca gtccaagttc atgataatga tgaaattatt 4140
tccagtgaca ttttgaacat gtaaactcat gaacatttac tattcattat tcaatgattt 4200
agcatttact cgttgaatga attgtgtatt caatttcttt gtcaagctca aaagcaccta 4260
gcgacatttt aaatcaattt ccttgtctat tcagtcattc cacaaattta agtgatcaaa 4320
acatcaatga acaataaaca ataccaatgg attttgtaag cagatttctt ttcatttcct 4380
gcttacacag acgacctcag tgcttttcag actattagct atcatcgtcc attttgcata 4440
gggttttgta agacccaaat gaaaagcatt cattttatca acagtttgcc ctacacagga 4500
ttatgtgtca acagagctat taatacacta aataatgaaa atcgtggcac ttaattagga 4560
aataggatta tccttttttt gcactggtga caattcaata aaagttgcaa ctgaactatt 4620
tacctgagta aaccagtcat ttactcaaat aagacactca cagattggct gtaaatcacc 4680
cgtacatttg tacataaaga gatattataa attacattgt ataatctcac agtaaagtca 4740
gtgagtcttg atattattaa agtgcgtgtg ataataatgt acccatttct tctgtgtgac 4800
taaagtcaat agaatgaccc aaaccagtaa gaattatacc tggaagacaa tattttcttc 4860
tttggtttgt taattttaag acaaagtata tacactatat cttgtatatc ctgtgaaatg 4920
ccatagtgga agtgcttaag caaaacagaa tattgtgtta aaagtgcaag aagcacagct 4980
tggaatatcc tgccataaat tgttaagtca ttgtgaggct taatgttttt tctaagtgtt 5040
cacctaataa tatgccattt actcaagagg cctactgcta caatatgtgt tatttccaaa 5100
aagtaacaaa attaaaggag tgttgcaggt cagaaagaaa gaacaaggag ctgctgatgt 5160
gacagcattc ctgacagaaa tttgcttttc tttgtcccta gcaaatgaaa aagtagaagc 5220
cataccagtg ccctagtttt gcagtaaaac aatgttatgg aaacagatac aacagttgcc 5280
atcagcaaaa cttagagcaa aggacataag gaccttttac aaaagaaact gaggatacag 5340
ctactctcag ctcaccttac aattgtgaat atagaataaa acttaaagac tggagcagaa 5400
actacatttt ttgaatgtct tcccaaggca ctgtagaatc cattcattac acaggatctc 5460
aatttcttca agtttagatc agcactgatg atgtttcttt ctgatgtagt tgattcggtg 5520
atatgtaaag attattacag ttttctctgt tgccttattt ctgtttcgac aggcaatgaa 5580
aatgaaaagt atttatttta ttgctggact ccttttaatg atagttcaag gcagctggca 5640
aaatcctctt caggatacag aggagaaatc aaggtaaatt ctttagatca acctcctaga 5700
aacccttagc ttgaactttg ctttcttatt gttgaaagca aagcatacca tgatttatag 5760
aaagcactta atctaccatt ggaactggta aaagctttta atgttgtgaa gtgtggcaca 5820
gtagcttttg gtattctaaa tagtaagtgc tttagttacc caataaacct tgtaagtgtg 5880
cagtatccaa gctacaatat tgttccattt ttattagcct gctaacaacc aaaactatgc 5940
cacatttttt cactttataa atgataatgt taaaagaaat gtagatatta caattattaa 6000
gtacaattaa atgtagaatt aaatttatta aatgtagaca ttctacacca gtagaatgta 6060
ctgtagtcat tgccagttaa caacttctaa acttttctta gatcattcaa agcttcccag 6120
tctgaaccat tagatgaatc taggcagctg aatgaagtaa agcgtcattc acaaggcaca 6180
ttcaccagtg actacagcaa gtacttggac agcagacgag ctcaggattt tgtgcaatgg 6240
ttaatgagca ctaaaagaaa tgggtaagcc tttttggtca cattgctcta tccacactga 6300
taaaacagcc aaaaatcaga actaattcaa tttctttatt tcagcttccc atcacagacc 6360
atatgtaatc ctcaaattac taatgtactt agtggaaatt tatatagaac tctaatatgt 6420
tattagaaat ggggaatatg tacaaaatta gtcaaaattg attggataaa ttagatgtag 6480
agtcattata ttatctagta tgcgacctaa aatgagactg tatctcagcc tctttgggac 6540
agatagctca ttcactgaaa caaatttcaa tatgtacaag gtacaccaat atagggtcaa 6600
aatgtagctg ctgggccttc atagaacctt ctattcatag ttgctctaat cctgaagttt 6660
catttgtgaa aagagagctg agatttaagc ttaattgacc aatggtcttc aagtgttctg 6720
ttaaggcatg agttggcatg gttttttgaa ggtacagttt gcttaaacaa aggctaacaa 6780
gctaagagga tgaataatga aatagcaagt aattctatgc cctatgttct ggagaagatt 6840
tgcccacaga atcaaaaaat ctgttatatg aatcttttaa atcactgtgg aaatctttaa 6900
agtaatcatt tctagatata gtattaattt aaatacataa gcctgaaaat atttataatg 6960
ttcattcatc atttgtggtt gtttatgata atacatagat aataaccaat agaattttat 7020
cagctcattc atgctctgca atgtactgct aataatgatc taatggttgt ggaattccac 7080
tacagccaac aaggacagga ggacaaagag aatgacaaat tcccggatca gctctcaagg 7140
tattctattt gttacaaact tgttactcaa aactaggttg agttaatttt ctgttaaaaa 7200
agaaataaat aaactggtat tctttaaaat gataacataa tcagagaaat acttaccaaa 7260
agaaattatg ctgcacctgt tctttttatc aggaaagaga aaacatctgc ttttgatgcc 7320
tgtacatgtg tgcttttgct atgtgcaatg acttcaagaa acgcaatagc atgagaataa 7380
tatataccag gaaaggacaa aaaaaaaaaa caaaaaacaa aaacctacag aaaaagctac 7440
aggaaaatca ttgtgttgtg agaaagaaaa caaattacaa cactaacaaa aggaaaatgg 7500
ccaggcacaa atttgtggag ggctgataaa acacaataca gtattcagcc ctcagcattc 7560
agtcccattt tatacaaatc tatcattaca gaaaaaaaaa aaaaaaagaa gaaagaaaaa 7620
gaaaaagaaa gaaagaaaag aagagaaaaa gaaaagcatt tgtgctgaat gcttaaattt 7680
tgtttcctgt gagtctagga tagaggtcaa ggagttggaa atttggaaaa aacaacagcc 7740
acaacaacaa atctctgttt gccaaacaga ttgaaatact tacacttatt ttttcttttg 7800
atatacttac acttattttt tctttttgat tacaaaaatt cttccattct aaacactatc 7860
aatataatac aaaatctttc agcttcagtg aaacagcata tcttaacaaa attaacattt 7920
tatctagtta acatgctgat gggaagtgca gatggtacta aattgggagg aatggtgaaa 7980
tcccaggaag acaaaaaaat aatacaaaac gctaaaggcc ctggaagttt ggcagaaatc 8040
agcaaactct gaaatagaaa ttcaagggaa cttttcatgg gaatatatcc caaaacactt 8100
cttcttctca atgcagcttg tcttcctaag tgttggtgtg acataatttt gctataaatt 8160
tgagaaagaa gcaggggtag gattgggaag caatttcttt tctattaagc tagagtttat 8220
caagtttgaa attgttccaa gattattact ttgttgaaaa caatggatat tcacaaaaca 8280
attcaaaatt aagcagagta atatcagaaa gataagtctg aggacgttgg ctgagaaata 8340
atgaagatga gcacagaagg agtagcttaa tggaattttt aattagtgca gttagaggta 8400
acaaaaagta tgtctgtaga atatggtagt ttactgtgca gagctaattc acctagcaga 8460
agaccgatcc agtgcagtta tacatgcttt cagtacctta tttgtttaga gctagttcag 8520
ttaatacaaa actgtactgt gtagcatata cgttgctttc agaactgaaa aatgaaaata 8580
tcaatagtaa tttgagcagc aaagaaaaac tctctctctg atgcagaatg tttagtggga 8640
aatttaaatt attaaatgct gaaaagatgt gataagtaat gcagtctgat cctagtttct 8700
tgtacctctt tcagtaatgc aatttccaag cgtcattctg aatttgagag acatgctgaa 8760
ggcacctaca ccagtgatat cacctcttat ttggaaggtc aagctgccaa agaattcatt 8820
gcttggttag tgaatggacg aggaagaaga gagtaagaat ccatccattc ctattattaa 8880
attttgcttt gctttcaagg ttagaaatca aatctgatga aatcttgatg tatttttgat 8940
cattcctact gtacttcacg ttttcctttt gtaatagaag atactgtgtg gctgttctta 9000
agttcaaaca tcttaagtta ttcaaacatc ttaagttatt aagttcaaac atagactgaa 9060
acaaacatgt tcagtatact gttacactct taaaggtcaa attatattta aggtcaatat 9120
atctaaaact aaaccagagt ttcctctaat taacaccttt tcctcatcac ataacattgc 9180
ttacagtctc cttaactctc acagagatga gtcatgttag tgtcgtctta aatttccctt 9240
tttctctgcc tctgtagtca atcatttcct ctgcaatatt tccatgatga aatttttaac 9300
aatgtcactg ccaccacagc cattttccat gctctggtga tcattcccag ttactgatgt 9360
cttcttttgc tcttcctgtt atccacaaag tctccacaaa ttgtagcatc aaaaataacc 9420
ataatttcct acagctttcc cttattttcc tttttaaagc acctctactt cttttgctat 9480
agttcacagt ttccaagact cagctccata gttttgattc aggtacatct cagacttttt 9540
cattatcttg ctctccatat attcagtgca aatttctcaa acctattcac ttcatctgct 9600
cacccatgat tttgtatctt tttcatgaga cttactcttg gaacagcctt aatttcaggc 9660
actttctgtc ttatcaccat tccaagtctg tgcagttagg agttacatca cacacacacc 9720
actctctacc ttttaactgt tctgtacttt ttgtgccttt agactgtaag ctcttcgggg 9780
caaggatttc agttcatcct attattatat gttttgctag ctgtaaagta ctatgtaaat 9840
ctctcatata aaaataataa ataacaataa caataaaata taatctataa gacaagattg 9900
tgtgttgtag tcaaggaaga ctaacaaggt aaaagtaaat tatgacaatg atgaaaaaca 9960
ctgctaagtg aatgaggtca agttgtacag ataaggaaaa ggctagtgcc acttccaggt 10020
gatgttcaga tccgcagcca ttttttctgt caccttttat tttcatgagc agagataggt 10080
ttgtaacttt caactgagct ctgatgatct gagcttaaaa accttaacaa accattacac 10140
acagagcatc ctcctacttc ctaatattta gtactgtttg cagtcagact tgaatgtagc 10200
ttttcctaaa attagtgcat aaaaaaaaca gtctgcctgt cgctgatctc agaacatagt 10260
gtgcctcaat cagtaaacct cactggaacc tagaaaaatc ttactttatt tggtggtttg 10320
aaacaagctt tctaaataaa ccccttggta aaatggcttt ccagtggtca gaaatagatg 10380
catcaagatt agcgatggga ttccaaatac aaaattctac tatgcaacaa aaaagcacat 10440
acatttaata cagatatttt aattcagtta ttttatcccc ccaaaaagct gctgctttta 10500
ctgattaaat acctatgcaa tacaattgta aatataaaac caggacttta agagtcaaat 10560
ggaaaaatct gtatgaaaat ttgactgtag tgttttaata gtgatatttc tttaaagaat 10620
gattcttagt aaatctactg tgagttttat tgatgactag tttacttggc aaatatgttg 10680
ttgacgtagt tatgcttttg catctatagc tttaaaaaaa tctgtgaaaa aacactaaag 10740
aggaatctag actagaggaa gactaaacga agagtccctt ctggtgaagg aagactttga 10800
tgctttcttc tgcagttaaa ctaatacttc ataagatgtt ctgatatgct tcatgctatg 10860
cagagcccta acatttggct caacctttaa ccctttaaaa tcaaaggaaa tattcagtaa 10920
ttaagtacct ataataaggc ccttttatca gtttgtttct ttggacttac aaattgtttc 10980
ataatttctt tcctttgaag tttccccgaa aaagctttaa tggctgaaga aatgggccga 11040
agacatgcgg atggcacctt cacaagtgat atcaacaaaa tcctggatga tatggctgcc 11100
aaagagttcc taaaatggct gattaacaca aaagttactc aaaggtgcct gaatttttat 11160
catatttttc ttaatatatt ggtttttttt tttaaatgca tttgtcacac atagggaaaa 11220
cacagctctc ttttctctta aatatagaaa tatgtgtaac agtgttacaa actttatgtt 11280
acagacagtg ctgaagaagg ctatgacact ctaaaaagaa gtgattccaa acagctactt 11340
aagaaccaca ctttaatttc tttgtatata gacactgttg ggaaaagaac attttctatt 11400
tgagctctac aaacaggtta atatttgtgg attatttttt gacaccgaca aagcgtttta 11460
aaactaatac agtgtccact taaggcacac gtgaaggcca gattttctaa aatgttgggt 11520
gcccattgca aaagtttgtt aactttgcct tttgacacct aaaggacgtc agatctctag 11580
ttaatctcgt aaatatgaaa cagcaaaaaa aattgaattt atagttaaaa catggatttg 11640
aataacaaat tcatattttt gtctttgttt ttggttaaga actgaaataa ttaatttaag 11700
aaagtcatct atttgaaact attaaaaatc ttttccttta ggaatagttt tcggtctttt 11760
attagagaga agcattaagg gatttgtgga actaatatct gctaattctt aacaagaaaa 11820
aatatatttc acacaaatgt gacaaatcat gtgagattta tattatgtcc taattgtttg 11880
gttttttttt ctccccagag accttttggg agaataccag taagaatgca ggataaaaat 11940
gggataaatg aaaatcttca ctgcctcaac tgccagctac aaagagattc tgaaatctat 12000
attgtcattt acattgggtg taacaaagct atgtcacctt gcatgccggg aaataattgt 12060
agtgtcattt aatgttgcca aaggattata tatggaatac ctatcatcta agggatggtt 12120
atcaaaatag atttaacatt attaaagttg cacttcttta tattttccac ttatttggac 12180
tgaaatagct ccatttatat ttacttatga aattattttt ctaaaaattg acttatgcac 12240
gcaaatgttt caatcaccag ttattatcta ggggctggca gctgcaaatg tctgaaaagt 12300
gaatatccct gttgatacct ttgttataaa aaggctaagc tttaagaata ttaaacaata 12360
gccttaaata aatttttttc aagcttcttt tctgttgact tttcatctgt atatcccttt 12420
tttctgtttc cactccattt taatttattc taatattatt gtcactgaga tcttggaact 12480
ggcttgttca gaatgataaa acaaaatcag cattttgtca attttctcta ttactatttt 12540
atgagaattg ctgtatataa agtttgatca tacttatctg ataaagaata atgtaagagg 12600
aagctagaga tggaagcaag actgaacagt attcattatc ataaattgct gtttctcaga 12660
gaagcaggag gacagatttg aaaaatatta accgaaattt acttcattaa tagtggaaac 12720
tctgccacct aattctgtca aggtttataa gactgctgag taaagccaac tcaaatggcc 12780
ctgagatcat tctaatgatc tatgttatat tcttttctgt attaaagatt cagttatata 12840
cagttggatc catatggtct aggctgaaat ggtaca 12876
<210> 2
<211> 34
<212> DNA
<213> 人工序列(Artificial)
<400> 2
caaaaaaaaa aaacaaaaaa caaaaaccta caga 34
<210> 3
<211> 33
<212> DNA
<213> 人工序列(Artificial)
<400> 3
tctgtaatga tagatttgta taaaatggga ctg 33
<210> 4
<211> 30
<212> DNA
<213> 人工序列(Artificial)
<400> 4
acaacactaa caaaaggaaa atggccaggc 30
Claims (3)
1.一种基因Gcg在检测家鸡源性成分中的应用，基因Gcg的核苷酸序列如SEQ ID NO.1所示。
2.一种用于检测家鸡源性成分的试剂盒，其特征在于，包括基因Gcg的特异性引物，特异性引物的核苷酸序列如SEQ ID NO.2和SEQ ID NO.3所示。
3.根据权利要求2所述用于检测家鸡源性成分的试剂盒，其特征在于，还包括探针，探针的核苷酸序列如SEQ ID NO.4所示。
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