CN101875975A - 一种鉴别羊肉和鸭肉的pcr-rflp方法 - Google Patents
一种鉴别羊肉和鸭肉的pcr-rflp方法 Download PDFInfo
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Abstract
羊肉和鸭肉鉴别的PCR-RFLP方法属于分子生物学检测领域。它设计了一种羊肉制品肉种来源鉴别的方法，解决了目前羊肉和鸭肉鉴别方法准确性低的缺陷，提供了鉴别羊肉和鸭肉的PCR-RFLP检测技术。可将羊肉和鸭肉按以下方法加以鉴别：提取样品DNA，采用通用引物扩增和凝胶电泳检测，然后分别用Bsu36I和SpeI限制性内切酶进行PCR-RFLP分析，即可区分羊肉和鸭肉。本发明采用PCR-RFLP方法，具有简单、快捷、廉价和准确性高的特点。
Description
技术领域
本发明属于分子生物学检测领域，涉及到羊肉和鸭肉的鉴别。
技术背景
羊肉中含有丰富的优质蛋白和大量的矿物质元素等，具有很高的营养价值。寒冬吃羊肉可益气补虚，促进血液循环，增强御寒能力。中医认为，羊肉还有补肾壮阳的作用，故而羊肉深受人们的青睐。然而近年来，一些不法商贩以鸭肉串当做羊肉串进行销售来欺骗消费者的事件屡有发生，这种以鸭肉替代羊肉进行销售的行为不仅给消费者带来损失，也给羊肉产业的健康发展造成不良影响。为了确保广大消费者的权益，为执法人员提供技术支撑，需要对羊肉和鸭肉进行准确的鉴别。
对生鲜肉进行感官鉴别时，根据一些物种肉在颜色、气味、滋味、纤维粗细等方面的特点差异，借助“看、闻、摸”手段，综合三个要点得出的判断，对肉类进行感官鉴别。这在实际应用中存在如下问题：对于经过加工的肉制品，由于加入色素、食品添加剂、香料等物质，通过感官手段难以进行准确鉴别。例如：制假者将鸭肉串刷上羊油，加上佐料等物质，靠传统的感官方法难以检验。
免疫学检测是基于可溶性抗原与抗体的免疫反应，应用种间特异性抗原进行设计研究的方法。GiovannacciI等2005年对动物物种检测的ELISA方法进行了全面综述，认为样品经过不同温度的加热处理会影响ELISA方法的检测准确性。特别是当样品经过高温处理后，蛋白发生了变性，改变了特异抗原决定簇，因此很难鉴别和区分动物物种。目前用于动物源性检测的试剂盒灵敏度较低，不适于推广使用。
PCR-RFLP技术快速、简便，在肉产品的种属鉴别方面已有相关报道，但未见有羊肉和鸭肉鉴别方面的报道。
发明内容
本发明的目的是解决目前羊肉和鸭肉鉴别方法准确性低的缺陷，提供一种羊肉和鸭肉鉴别的PCR-RFLP方法，为打击鸭肉代替羊肉销售的不法行为提供技术支撑。
可将羊肉和鸭肉按以下方法进行鉴别：一、将取50mg待检样品放入1.5mL的EP管(EP管应高压灭菌)中，用小剪刀将其剪碎。二、加入STE抽提液600μL、ProK(20mg/mL)20μL及20％的SDS溶液15μL，在震荡器上充分混匀。三、将EP管置入水浴锅，55℃消化电泳图。三个图中的“阴”为阴性对照样品，M为分子量内标。
具体实施方式
本实施方式一：一、将取50mg待检样品放入1.5mL的EP管(EP管应高压灭菌)中，用小剪刀将其剪碎。二、加入STE抽提液600μL、ProK(20mg/mL)20μL及20％的SDS溶液15μL，在震荡器上充分混匀。三、将EP管置入水浴锅，55℃消化过夜(12-16h)，在此期间可取出颠倒混匀几次，以便消化彻底。四、将EP管取出，每管加入600μL的Tris饱和酚，轻轻摇动混匀15min，然后在12000rpm离心10min。五、转移上清液至新的EP管，加入等体积的酚氯仿异戊醇混合液(酚∶氯仿∶异戊醇体积比为25∶24∶1)，颠倒混匀10min，然后12000rpm离心10min。六、转移上清液至新的EP管，加入等体积的氯仿异戊醇混合液(氯仿∶异戊醇体积比为24∶1)，轻轻混匀10min，然后在12000rpm离心10min。七、转移上清液至新的EP管，先加入1mL冰乙醇，再加入60μL的NaAc(3M)，轻轻水平摇动，可见絮状、白色DNA出现。将EP管放入-20℃冰箱冷冻30min，然后在12000rpm离心10min，可见DNA沉于管底。弃无水乙醇，再用75％的乙醇洗涤DNA两次，然后在12000rpm离心10min；自然干燥后，加入50μL TE溶液溶解。八、PCR扩增及检测：引物用可扩增所有脊椎动物Cytb序列的通用上游引物F：5’-TAC CAT GAG GAC AAA TAT CAT TCT G-3’和通用下游引物R：5’-CCT CCT AGT TTG TTA GGG ATT GAT CG-3’。PCR反应总体积为25μL，其中10×PCR Buffer 2.5μL，Taq DNA聚合酶0.2μL(2.5U/μL)，dNTPs(2.5mmol/L)2μL，10pmol/μL的上、下游引物各2μL，模板DNA 250ng，灭菌蒸馏水补足总体积。扩增条件为：95℃变性10min，95℃变性45s，53℃退火60s，72℃延伸60s，35个循环后在72℃继续延伸7min。取4μL的PCR产物与1μL的6×loadding buffer混合，采用TAE缓冲系统，2％琼脂糖凝胶(加入染色剂Gold view I)在5v/cm恒压电泳条件下约25min，在凝胶成像系统中观察并记录。九、Bsu36I酶切分析：对步骤八中的PCR产物用限制性内切酶Bsu36I进行PCR-RFLP分析；Bsu36I只能将鸭的PCR产物切割为95bp和377bp的片段，而不能切割山羊和绵羊的PCR产物，根据琼脂糖凝胶检测结果是否可检测到95bp和377bp的条带来判断是否为鸭肉。酶切体系为20μL，其中：DNA(PCR产物)1μg，10×NEB buffer 2μL，100×BSA0.2μL，Bsu36I限制性内切酶(10000U/mL)0.5μL，灭菌蒸馏水补足体积。混匀并离心后在37℃消化16h，然后在65℃处理20min灭活内切酶活性，取7μLPCR产物与1μL 6×loadding buffer混合，采用TAE缓冲系统，2％琼脂糖凝胶(加入Gold view I染色剂)在5v/cm恒压电泳条件下约25min，在凝胶成像系统中观察并记录。十、SpeI酶切分析：对步骤八中的PCR产物用限制性内切酶SpeI进行PCR-RFLP分析；SpeI可将绵羊和山羊的PCR产物切割为326过夜(12-16h)，在此期间可取出颠倒混匀几次，以便消化彻底。四、将EP管取出，每管加入600μL的Tris饱和酚，轻轻摇动混匀15min，然后在12000rpm离心10min。五、转移上清液至新的EP管，加入等体积的酚氯仿异戊醇混合液(酚∶氯仿∶异戊醇体积比为25∶24∶1)，颠倒混匀10min，然后12000rpm离心10min。六、转移上清液至新的EP管，加入等体积的氯仿异戊醇混合液(氯仿∶异戊醇体积比为24∶1)，轻轻混匀10min，然后在12000rpm离心10min。七、转移上清液至新的EP管，先加入1mL冰乙醇，再加入60μL的NaAc(3M)，轻轻水平摇动，可见絮状、白色DNA出现。将EP管放入-20℃冰箱冷冻30min，然后在12000rpm离心10min，可见DNA沉于管底。弃无水乙醇，再用75％的乙醇洗涤DNA两次，然后在12000rpm离心10min；自然干燥后，加入50μL TE溶液溶解。八、PCR扩增及检测：引物用可扩增所有脊椎动物Cyt b序列的通用上游引物F：5’-TAC CAT GAG GAC AAA TAT CATTCT G-3’和通用下游引物R：5’-CCT CCT AGT TTG TTA GGG ATT GAT CG-3’。PCR反应总体积为25μL，其中10×PCR Buffer 2.5μL，Taq DNA聚合酶0.2μL(2.5U/μL)，dNTPs(2.5mmol/L)2μL，10pmol/μL的上、下游引物各2μL，模板DNA 250ng，灭菌蒸馏水补足总体积。扩增条件为：95℃变性10min，95℃变性45s，53℃退火60s，72℃延伸60s，35个循环后在72℃继续延伸7min。取4μL的PCR产物与1μL的6×loadding buffer混合，采用TAE缓冲系统，2％琼脂糖凝胶(加入染色剂Gold view I)在5v/cm恒压电泳条件下约25min，在凝胶成像系统中观察并记录。九、Bsu36I酶切分析：对步骤八中的PCR产物用限制性内切酶Bsu36I进行PCR-RFLP分析；Bsu36I只能将鸭的PCR产物切割为95bp和377bp的片段，而不能切割山羊和绵羊的PCR产物，根据琼脂糖凝胶检测结果是否可检测到95bp和377bp的条带来判断是否为鸭肉。十、SpeI酶切分析：对步骤八中的PCR产物用限制性内切酶SpeI进行PCR-RFLP分析；SpeI可将绵羊和山羊的PCR产物切割为326bp和146bp，但不能切割鸭的PCR产物，根据琼脂糖凝胶检测结果是否可检测到326bp和146bp的条带来判断是否为羊肉。
本发明在鉴别过程中采用PCR-RFLP方法，具有简便、快捷、廉价和准确性高的特点。
附图说明
图1是具体实施方式一中步骤八得到的绵羊(S)、山羊(G)和鸭(D)PCR产物检测的凝胶电泳图；图2是具体实施方式一中步骤九用Bsu36I限制内切酶酶切对具体实施方式一中步骤八得到的绵羊(S)、山羊(G)和鸭(D)的PCR产物进行PCR-RFLP分析后检测的凝胶电泳图；图3是具体实施方式一中步骤十用SpeI限制性内切酶酶切对具体实施方式一中步骤八得到的绵羊(S)、山羊(G)和鸭(D)的PCR产物进行PCR-RFLP分析后检测的凝胶bp和146bp，但不能切割鸭的PCR产物，根据琼脂糖凝胶检测结果是否可检测到326bp和146bp的条带来判断是否为羊肉。酶切体系为20μL，其中：DNA(PCR产物)1μg，10×NEBbuffer 2μL，100×BSA 0.2μL，SpeI限制性内切酶(10000U/mL)0.5μL，灭菌蒸馏水补足体积。混匀并离心后在37℃消化16h，然后在65℃处理20min灭活内切酶活性，取7μL PCR产物与1μL 6×loadding buffer混合，采用TAE缓冲系统，2％琼脂糖凝胶(加入Gold view I染色剂)在5v/cm恒压电泳条件下约25min，在凝胶成像系统中观察并记录。
附：鉴别绵羊、山羊和鸭肉所用限制性内切酶的酶切位点
限制性内切酶Bsu36I(CC↓TNAGG)
限制性内切酶SpeI(A↓CTAGT)
附：溶液配制表
STE抽提液：10mL Tris-HCl(1M；pH 8.0)，2mL EDTA(0.5M；pH 8.0)，100mL NaAc(1M)，888mL水，高压灭菌。
20％SDS：200g SDS溶于900mL水中，用盐酸调pH值至7.2，加水定容至1000mL。
1M Tris-HCl：121.4g Tris溶于800mL双蒸水中，用盐酸调pH值至8.0，定容至1000mL，高压灭菌。
TE缓冲液(pH 8.0)：1mL Tris-HCl(1M；pH 8.0)，0.1mL EDTA(0.5M；pH 8.0)，加双蒸水定容至50mL，高压灭菌。
20mg/mL ProK：100mg蛋白酶K溶于5mL灭菌双蒸水，-20℃分装保存。
TE缓冲液(pH 8.0)：1mL Tris-HCl(1M；pH 8.0)，0.1mL EDTA(0.5M；pH 8.0)，加双蒸水定容至50mL，高压灭菌。
Claims (5)
1.一种鉴别羊肉和鸭肉的PCR-RFLP方法，其特征在于可将羊肉和鸭肉按以下方法加以鉴别：一、将取50mg待检样品放入1.5mL的EP管(EP管应高压灭菌)中，用小剪刀将其剪碎。二、加入STE抽提液600μL、ProK(20mg/mL)20μL及20％的SDS溶液15μL，在震荡器上充分混匀。三、将EP管置入水浴锅，55℃消化过夜(12-16h)，在此期间可取出颠倒混匀几次，以便消化彻底。四、将EP管取出，每管加入600μL的Tris饱和酚，轻轻摇动混匀15min，然后在12000rpm离心10min。五、转移上清液至新的EP管，加入等体积的酚氯仿异戊醇混合液(酚∶氯仿∶异戊醇体积比为25∶24∶1)，颠倒混匀10min，然后12000rpm离心10min。六、转移上清液至新的EP管，加入等体积的氯仿异戊醇混合液(氯仿∶异戊醇体积比为24∶1)，轻轻混匀10min，然后在12000rpm离心10min。七、转移上清液至新的EP管，先加入1mL冰乙醇，再加入60μL的NaAc(3M)，轻轻水平摇动，可见絮状、白色DNA出现。将EP管放入-20℃冰箱冷冻30min，然后在12000rpm离心10min，可见DNA沉于管底。弃无水乙醇，再用75％的乙醇洗涤DNA两次，然后在12000rpm离心10min；自然干燥后，加入50μL TE溶液溶解。八、PCR扩增及检测：引物用可扩增所有脊椎动物Cyt b序列的通用上游引物F：5’-TAC CAT GAG GAC AAA TAT CAT TCT G-3’和通用下游引物R：5’-CCT CCT AGT TTG TTA GGG ATT GAT CG-3’。PCR反应总体积为25μL，其中10×PCR Buffer 2.5μL，Taq DNA聚合酶0.2μL(2.5U/μL)，dNTPs(2.5mmol/L)2μL，10pmol/μL的上、下游引物各2μL，模板DNA 250ng，灭菌蒸馏水补足总体积。扩增条件为：95℃变性10min，95℃变性45s，53℃退火60s，72℃延伸60s，35个循环后在72℃继续延伸7min。取4μL的PCR产物与1μL的6×loadding buffer混合，采用TAE缓冲系统，2％琼脂糖凝胶(加入染色剂Gold view I)在5v/cm恒压电泳条件下约25min，在凝胶成像系统中观察并记录。九、Bsu36I酶切分析：对步骤八中的PCR产物用限制性内切酶Bsu36I进行PCR-RFLP分析；Bsu36I只能将鸭的PCR产物切割为95bp和377bp的片段，而不能切割山羊和绵羊的PCR产物，根据琼脂糖凝胶检测结果是否可检测到95bp和377bp的条带来判断是否为鸭肉。十、SpeI酶切分析：对步骤八中的PCR产物用限制性内切酶SpeI进行PCR-RFLP分析；SpeI可将绵羊和山羊的PCR产物切割为326bp和146bp，但不能切割鸭的PCR产物，根据琼脂糖凝胶检测结果是否可检测到326bp和146bp的条带来判断是否为羊肉。
2.根据权利要求1所述的一种鉴别羊肉和鸭肉的PCR-RFLP方法，其特征在于步骤二用STE抽提液。
3.根据权利要求1所述的一种鉴别羊肉和鸭肉的PCR-RFLP方法，其特征在于步骤八中扩增引物为用通用上游引物F：5’-TAC CAT GAG GAC AAA TAT CAT TCT G-3’和通用下游引物R：5’-CCT CCT AGT TTG TTA GGG ATT GAT CG-3’。
4.根据权利要求1所述的一种鉴别羊肉和鸭肉的PCR-RFLP方法，其特征在于步骤九用限制性内切酶Bsu36I进行PCR-RFLP分析。
5.根据权利要求1所述的一种鉴别羊肉和鸭肉的PCR-RFLP方法，其特征在于步骤十用限制内切酶SpesI进行PCR-RFLP分析。
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