食品中动物源性成分定性鉴定方法研究
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摘要
本研究采用高度特异引物PCR法、基因条形编码法(DNA barcoding)、限制性内切酶酶切片段长度多态性法(PCR-RFLP)、通用引物-聚合酶链式反应-限制性片段末端长度多态性法(UP-PCR-TRFLP)对食品中猪、牛、羊源性成分进行检测,比较四种方法各自的优缺点及适用范围,建立食品中动物源性成分定性鉴定方法。主要研究结果如下:
(1)试验证实:基于线粒体DNA(mtDNA)D-loop区和细胞色素b(Cytb)基因设计出的引物具有高度特异性,能够对食品中猪、牛、羊源性成分进行准确的定性检测,且能够检测混合样品。
(2)利用双脱氧法sanger法对COⅠ基因测序并对测序结果进行多重序列比对,发现猪、羊、牛COⅠ基因的碱基差异为19%～25.9%,物种内的碱基差异仅为0.1%～1.6%。COⅠ基因含有大量的具有物种特征的单核苷酸位点,因此,利用猪、牛、羊COⅠ基因的碱基差基因条码技术能够非常准确地鉴定食品中的猪、牛、羊3种动物源性成分,但是受sanger法测序技术的限制,该方法不适于混合样本的检测。
(3)利用PCR-RFLP法对猪、羊、牛3个物种的COⅠ基因进行酶切,经HinfⅠ酶切后均可获得3个物种特征片段长度段,猪的片段长度分别为40bp、411bp、449 bp;牛分别为46p,377bp,479 bp;羊分别为144bp、376bp~381 bp。通过电泳检测, PCR-RFLP法能够对食品中的猪、牛、羊3种动物源性成分进行定性鉴定,但不适于混合样品的检测。
(4)采用UP-PCR-TRFLP法在COⅠ基因保守区设计一对牛羊通用引物,并对上游引物的5’端用6-FAM进行荧光标记,在下游引物的5’端用ROX进行荧光标记,然后用限制性内切酶HinfⅠ和BlnⅠ进行酶切消化,再经毛细管电泳建立牛和羊的特有峰谱。牛的PCR产物经过限制性内切酶HinfⅠ酶切后在上游会产生373bp长度的片段,在下游产生46bp长度的片段,这两个片段为牛酶切后的特征片段。羊的PCR产物经过限制性内切酶BlnⅠ酶切后在上游会产生515bp长度的片段,在下游产生203bp长度的片段,这两个片段为羊酶切后的特征片段。不同物种可产生不同的峰谱。因此该法可用于牛、羊肉的定性鉴定分析,且能够用于混合样本的的鉴定。
(5)mtDNA上的COⅠ基因测序分析和限制性内切酶切分析结果均表明:COⅠ基因含有大量能够用于物种鉴定的碱基突变,在猪、牛、羊的物种鉴定中,具有很好的区分鉴别能力,可以应用于食品中动物源性成分的鉴定工作。
The animal-derived ingredients from pork, beef, mutton and chevon in foods were identified by molecular biology technology such as species-specific polymerase chain reaction(PCR), DNA barcoding, restriction fragment length polymorphism of polymerase chain reaction products(PCR-RFLP), terminal restriction fragment length polyraorphism by universal primer polymerase chain reaction (UP-PCR-TRFLP) to compare advantages and disadvantages of the four methods , and to establishment the qualitative identification methods for animal-derived ingredients in foods. The results were as follows:
(1) The tests showed that the gene specific primer designed according to the mtDNA D-loop region and Cytb genes were of high specificity. The any animal-derived ingredients from pork, beef, mutton and chevon in foods could be identified accurately by the specific primer, it also could be used to detect mixed samples.
(2)After the COⅠgene had been sequenced by deoxidizing method, the sequencing results were compared by multiple sequence alignment, it was found that the nucleotide difference of COⅠgenes between swine, sheep, cattle are 19%～25.9%, nucleotide differences within species are only 0.1%～1.6%. COⅠgene Contained a large number of single nucleotide mutation sites which can mark species characteristics, so the animal-derived ingredients from pigs, cattle and sheep products in food could be identified accurately by gene barcode technology on bases difference of COⅠgene, but this method was not suitable for the detection of mixed samples.
(3)Three specific gene segments of pork, beef and mutton were obtained after COI genes had been identified by Hinf I restriction enzymes among enzyme digestion analysis PCR-RFLP. The lengthes of segments were 40bp, 411bp and 449bp for pork, 46bp, 377 bp and 479bp for beef , 144bp, 376~381bp for sheep respectively. Based on the electrophoresis test results, it was safe to say that PCR-RFLP method could be used for qualitative identification of animal-derived ingredients from pork, beef, mutton in food, but it was not suitable for the detection of mixed samples.
(4)A pair of universal primers for beef and mutton was be designed in conservative regions of COⅠg ene with the UP-PCR-TRFLP method . The forward PCR primer was labeled with 6-carboxyfluorescein amino hexy(6-FAM) fluorescent dye and reverse PCR primer was labeled with ROX fluorescent dye after digestion by restriction enzyme HinfⅠand BlnⅠ, The restricition fragment were analysed by capillary electrophoresis using an antomated DNA sequencer. The special peaks of swine and beef were established. The PCR products of beef could appeared 373bp fragments at upstream and 46bp fragments at downstream by the restriction enzyme HinfⅠ. Both of fragments were regarded as the characteristic fragments for beef. PCR products of sheep appeared 515bp fragments at upstream and 203bp fragments at downstream by same method. Both of them were regarded as the characteristic fragments for sheep. There were different electrophoresis peak spectrum from different species. Therefore, UP-PCR-TRFLP method could be used for identification of animal-derived ingredients from beef and mutton products in foods and it was also a detection method for mixed samples.
(5) The sequencing results of COⅠgene in mtDNA and extinction enzymes cut analysis showed that COⅠgene contained a large number of base mutation single nucleotide mutations which could be used for identification of species. Those mutation sites were of high distinguish function on species identification of pork, beef and mutton, so they could be used for identification of animal-derived ingredients in animal-derived ingredients
(1)试验证实:基于线粒体DNA(mtDNA)D-loop区和细胞色素b(Cytb)基因设计出的引物具有高度特异性,能够对食品中猪、牛、羊源性成分进行准确的定性检测,且能够检测混合样品。
(2)利用双脱氧法sanger法对COⅠ基因测序并对测序结果进行多重序列比对,发现猪、羊、牛COⅠ基因的碱基差异为19%～25.9%,物种内的碱基差异仅为0.1%～1.6%。COⅠ基因含有大量的具有物种特征的单核苷酸位点,因此,利用猪、牛、羊COⅠ基因的碱基差基因条码技术能够非常准确地鉴定食品中的猪、牛、羊3种动物源性成分,但是受sanger法测序技术的限制,该方法不适于混合样本的检测。
(3)利用PCR-RFLP法对猪、羊、牛3个物种的COⅠ基因进行酶切,经HinfⅠ酶切后均可获得3个物种特征片段长度段,猪的片段长度分别为40bp、411bp、449 bp;牛分别为46p,377bp,479 bp;羊分别为144bp、376bp~381 bp。通过电泳检测, PCR-RFLP法能够对食品中的猪、牛、羊3种动物源性成分进行定性鉴定,但不适于混合样品的检测。
(4)采用UP-PCR-TRFLP法在COⅠ基因保守区设计一对牛羊通用引物,并对上游引物的5’端用6-FAM进行荧光标记,在下游引物的5’端用ROX进行荧光标记,然后用限制性内切酶HinfⅠ和BlnⅠ进行酶切消化,再经毛细管电泳建立牛和羊的特有峰谱。牛的PCR产物经过限制性内切酶HinfⅠ酶切后在上游会产生373bp长度的片段,在下游产生46bp长度的片段,这两个片段为牛酶切后的特征片段。羊的PCR产物经过限制性内切酶BlnⅠ酶切后在上游会产生515bp长度的片段,在下游产生203bp长度的片段,这两个片段为羊酶切后的特征片段。不同物种可产生不同的峰谱。因此该法可用于牛、羊肉的定性鉴定分析,且能够用于混合样本的的鉴定。
(5)mtDNA上的COⅠ基因测序分析和限制性内切酶切分析结果均表明:COⅠ基因含有大量能够用于物种鉴定的碱基突变,在猪、牛、羊的物种鉴定中,具有很好的区分鉴别能力,可以应用于食品中动物源性成分的鉴定工作。
The animal-derived ingredients from pork, beef, mutton and chevon in foods were identified by molecular biology technology such as species-specific polymerase chain reaction(PCR), DNA barcoding, restriction fragment length polymorphism of polymerase chain reaction products(PCR-RFLP), terminal restriction fragment length polyraorphism by universal primer polymerase chain reaction (UP-PCR-TRFLP) to compare advantages and disadvantages of the four methods , and to establishment the qualitative identification methods for animal-derived ingredients in foods. The results were as follows:
(1) The tests showed that the gene specific primer designed according to the mtDNA D-loop region and Cytb genes were of high specificity. The any animal-derived ingredients from pork, beef, mutton and chevon in foods could be identified accurately by the specific primer, it also could be used to detect mixed samples.
(2)After the COⅠgene had been sequenced by deoxidizing method, the sequencing results were compared by multiple sequence alignment, it was found that the nucleotide difference of COⅠgenes between swine, sheep, cattle are 19%～25.9%, nucleotide differences within species are only 0.1%～1.6%. COⅠgene Contained a large number of single nucleotide mutation sites which can mark species characteristics, so the animal-derived ingredients from pigs, cattle and sheep products in food could be identified accurately by gene barcode technology on bases difference of COⅠgene, but this method was not suitable for the detection of mixed samples.
(3)Three specific gene segments of pork, beef and mutton were obtained after COI genes had been identified by Hinf I restriction enzymes among enzyme digestion analysis PCR-RFLP. The lengthes of segments were 40bp, 411bp and 449bp for pork, 46bp, 377 bp and 479bp for beef , 144bp, 376~381bp for sheep respectively. Based on the electrophoresis test results, it was safe to say that PCR-RFLP method could be used for qualitative identification of animal-derived ingredients from pork, beef, mutton in food, but it was not suitable for the detection of mixed samples.
(4)A pair of universal primers for beef and mutton was be designed in conservative regions of COⅠg ene with the UP-PCR-TRFLP method . The forward PCR primer was labeled with 6-carboxyfluorescein amino hexy(6-FAM) fluorescent dye and reverse PCR primer was labeled with ROX fluorescent dye after digestion by restriction enzyme HinfⅠand BlnⅠ, The restricition fragment were analysed by capillary electrophoresis using an antomated DNA sequencer. The special peaks of swine and beef were established. The PCR products of beef could appeared 373bp fragments at upstream and 46bp fragments at downstream by the restriction enzyme HinfⅠ. Both of fragments were regarded as the characteristic fragments for beef. PCR products of sheep appeared 515bp fragments at upstream and 203bp fragments at downstream by same method. Both of them were regarded as the characteristic fragments for sheep. There were different electrophoresis peak spectrum from different species. Therefore, UP-PCR-TRFLP method could be used for identification of animal-derived ingredients from beef and mutton products in foods and it was also a detection method for mixed samples.
(5) The sequencing results of COⅠgene in mtDNA and extinction enzymes cut analysis showed that COⅠgene contained a large number of base mutation single nucleotide mutations which could be used for identification of species. Those mutation sites were of high distinguish function on species identification of pork, beef and mutton, so they could be used for identification of animal-derived ingredients in animal-derived ingredients
引文
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