CN104017882B - 检测奶牛子宫内膜细胞炎性反应的荧光定量pcr试剂盒及其检测方法和应用 - Google Patents

检测奶牛子宫内膜细胞炎性反应的荧光定量pcr试剂盒及其检测方法和应用 Download PDF

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CN104017882B
CN104017882B CN201410270225.9A CN201410270225A CN104017882B CN 104017882 B CN104017882 B CN 104017882B CN 201410270225 A CN201410270225 A CN 201410270225A CN 104017882 B CN104017882 B CN 104017882B
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张世栋
王东升
董书伟
严作廷
王旭荣
杨峰
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Guangzhou peptide Agriculture Technology Co., Ltd.
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Abstract

本发明提供了一种检测奶牛子宫内膜细胞炎性反应的荧光定量PCR试剂盒,包括PCR缓冲液、TaqDNA聚合酶混合物,SYBR?Green染料,两对引物:引物1:上游引物为5’-TATGACCAGGGACCAGGTAC-3’,下游引物为5’-CTCTGCCCTTAGGGACTCACAG-3’;引物2:上游引物为5’-ATCGGCAATGAGCGGTTCC-3’,下游引物为5’-GTGTTGGCGTAGAGGTCCTTG-3’。本发明的试剂盒可以简便、快速,且能够以相对定量检测奶牛子宫内膜细胞中淀粉样蛋白(SAA)基因的转录水平,从而为快速判断奶牛是否患有隐性子宫内膜炎提供参考。

Description

检测奶牛子宫内膜细胞炎性反应的荧光定量PCR试剂盒及其检测方法和应用
技术领域
本发明属于PCR扩增技术领域,具体涉及检测奶牛子宫内膜细胞炎性反应的特异性荧光定量PCR试剂盒和检测方法。
背景技术
聚合酶链式反应(PolymeraseChainReaction,PCR)是DNA体外操作技术中最常用的技术,实现了靶DNA片段在反应液中呈指数倍扩增。荧光定量PCR技术结合了实时荧光检测技术和计算机分析技术,在PCR反应体系中加入特定的荧光标记物质,通过检测每个热循环后PCR反应体系中的荧光值的变化情况来实时监测DNA的扩增情况,并获得每个样本的荧光定量变化曲线,进而获得每个反应管中荧光变化达到阈值时的PCR循环数(Ct);以起始模板数的对数为横坐标,以Ct值为纵坐标制备标准曲线,据此各个标本的Ct值对每个反应的起始DNA模板数进行定量测定。
SYBRGreen是一种结合于双链DNA小沟中的荧光染料。与双链DNA结合后,其荧光发光度大大增强。这种性质用于扩增产物的检测非常理想。在PCR反应体系中,加入过量的SYBRGreen荧光染料能充分地、特异性地掺入DNA双链后,发射荧光信号,而不掺入链中的SYBR染料分子不会发射任何荧光信号,从而保证荧光信号的增加与PCR产物的增加完全同步。SYBRGreen在核酸的实时检测方面有很多优点,由于它与所有的双链DNA相结合,不必因为模板不同而特别定制,因此设计的程序通用性好,且价格相对较低。此外,由于一个PCR产物可以与多分子的染料结合,因此SRYBGreen的灵敏度很高。但是,由于SYRBGreen与所有的双链DNA相结合,因此由引物二聚体、单链二级结构以及错误的扩增产物引起的假阳性会影响定量的精确性。通过测量升高温度后荧光的变化可以帮助降低非特异产物的影响。通过熔解曲线来分析产物的均一性有助于分析由SYBRGreen得到定量结果的准确性。
奶牛隐性子宫内膜炎是产后恢复后普遍存在的一种产科疾病,由于无明显的临床症状,有关奶牛隐性子宫内膜炎的体外诊断也较为困难,该病往往对养殖经济造成巨大损失。
发明内容
本发明提供了一种体外分离的检测奶牛子宫内膜细胞炎性反应的荧光定量PCR试剂盒,利用该试剂盒进行的体外检测的结果可以为判断奶牛是否患有隐性子宫内膜炎提供较好的参考价值。为此,本发明还提供其检测方法和应用。
奶牛隐性子宫内膜炎是子宫上皮细胞的慢性炎症过程,而我们的研究结果表明,奶牛子宫内膜上皮细胞中淀粉样蛋白(SAA)基因的表达水平升高与子宫内膜炎直接相关。因此,本发明提供一种操作简便、快速,且能够以相对定量检测不同奶牛子宫内膜细胞中淀粉样蛋白基因(SAA)的相对转录水平(与ACTB相比较所得比值)的试剂盒,从而判断奶牛是否患有隐性子宫内膜炎。ACTB基因是一种看家基因,在细胞内表达稳定,是理想的内参基因。
本发明提供一种牛子宫内膜细胞炎性反应检测的荧光定量PCR试剂盒,包括含SYBRGreen染料的PCR反应液、TaqDNA聚合酶混合物和两对引物,所述引物序列如下:
引物1(SAA基因):上游引物序列为5’-TATGACCAGGGACCAGGTAC-3’,下游引物序列为5’-CTCTGCCCTTAGGGACTCACAG-3’;
引物2(ACTB基因):上游引物序列为5’-ATCGGCAATGAGCGGTTCC-3’,下游引物序列为5’-GTGTTGGCGTAGAGGTCCTTG-3’。
其中,所述牛为奶牛。
本发明的第二个目的是提供一种奶牛子宫内膜细胞炎性反应的相对定量检测方法,是以奶牛子宫内膜细胞中总mRNA反转录的cDNA为模板,利用引物1、2和SYBRGreen染料进行实时荧光定量PCR,根据扩增曲线判定结果:当引物1扩增的基因表达量相对于引物2扩增的基因表达量>5倍,则判断为奶牛隐性子宫内膜炎为阳性;反之则为阴性。
优选地,所述实时荧光定量PCR的反应条件为:95℃预变性2min;PCR反应39个循环,其循环过程为95℃变性20s,60.5℃退火30s,72℃延伸30s,每个循环后检测荧光信号值。
本发明还提供上述试剂盒在离体奶牛子宫内膜细胞炎性反应检测和鉴定奶牛隐性子宫内膜炎中的应用。
本发明提供的检测试剂盒和检测方法操作简便、快速,且能够以相对定量检测奶牛子宫内膜细胞中淀粉样蛋白(SAA)基因的转录水平,从而在体外条件下为快速判断奶牛的隐性子宫内膜炎提供参考,为疾病诊断、预防和提高经济效益提供帮助。本发明试剂盒的应用在有关奶牛隐性子宫内膜炎的体外实验研究和诊断中都具有重要的参考价值。
附图说明
附图用来提供对本发明的进一步理解,并且构成说明书的一部分,与本发明的实施例一起用于解释本发明,并不构成对本发明的限制。在附图中:
图1:引物1对奶牛SAA基因的检测结果。左图为扩增曲线,横坐标为PCR循环次数,纵坐标为相对荧光值(RelativeFluorescenceUnits,RFU);右图为溶解曲线,横坐标为温度,纵坐标为单位温度下相对荧光值的变化量。
图2:引物2对牛ACTB基因的检测结果。左图为扩增曲线,横坐标为PCR循环次数,纵坐标为相对荧光值(RelativeFluorescenceUnits,RFU);右图为溶解曲线,横坐标为温度,纵坐标为单位温度下相对荧光值的变化量。
具体实施方式
以下的实施例便于更好地理解本发明,但并不限定本发明。下述实施例中的实验方法,如无特殊说明,均为常规方法。下述实施例中所用的试验材料,如无特殊说明,均为自常规生化试剂商店购买得到的。
本发明的奶牛隐性子宫内膜炎的荧光定量PCR诊断试剂盒包括有:反转录液、2×SYBRGREEN预混液、引物混合液1、引物混合液2、mRNA提取液和超纯水。
试剂盒中各组成成分如下:
反转录液:含有DNA酶(25U/μL),RNase抑制剂(50U/μL),逆转录酶SuperScriptⅡ(250U/μL)。
2×SYBRGreen预混液:含有Taq脱氧核糖核酸聚合酶(0.1U/μL)、dNTPs底物(0.4mmol/L)、Mg2+(5.0mmol/L)、KCl(100mmol/L)、Tris·HCl(20mmol/L,pH8.3)、SYBRGreen染料。
引物混合液1(对奶牛SAA基因进行扩增):上游引物为10μmol/L、下游引物为10μmol/L,上游引物1序列为5’-TATGACCAGGGACCAGGTAC-3’,下游引物序列为5’-CTCTGCCCTTAGGGACTCACAG-3’;
引物混合液2(对奶牛ACTB基因进行扩增):上游引物为10μmol/L、下游引物为10μmol/L,上游引物1序列为5’-ATCGGCAATGAGCGGTTCC-3’,下游引物序列为5’-GTGTTGGCGTAGAGGTCCTTG-3’;
mRNA提取液:Trizol试剂,氯仿,异丙醇,乙醇(75%),4种试剂均分别包装。
超纯水:纯度超过18.25MΩ·CM。
一、提取mRNA:使用时,首先用细胞刷(母牛专用,国内外均有专利产品)从母牛子宫腔内取得子宫内膜细胞,将取得的细胞经生理盐水常规漂洗2次后,800rpm离心5min以收集细胞(>100个),加入0.5mlTrizol试剂,室温放置5min;再加入0.25ml氯仿,混匀后静置5min,12000g离心10min后,取上清液0.2ml至新的离心管,并加入异丙醇0.2ml,轻轻震荡混匀,静置5min后12000g离心10min,转移0.2ml上清液至新的离心管中,并加入乙醇,12000g离心5min,弃上清,加入20μL纯水溶解沉淀形成mRNA溶液。
二、反转录:取2μLmRNA溶液测定含量后,取200ngmRNA至PCR管中,加入4μL反转录液,并补加纯水至总体积10μL,然后进行反转录反应,所进行程序为:42℃加热15min,85℃灭活5s,再降温至4℃。
三、PCR反应:取出PCR管,并补加SYBRGreen预混液25μL,引物混合液(1或2分别在不同反应管)2μL,纯水13μL。将总体积50μL的PCR反应管放入荧光定量PCR仪,反应程序为:95℃预变性2min;PCR反应39个循环,其循环过程为95℃变性20s,60.5℃退火30s,72℃延伸30s,每个循环后检测荧光信号值。PCR扩增反应结束后,产物进行溶解曲线检测,程序为:55℃~95℃,0.5℃/10s。
结果计算与判断:扩增曲线结果表明SAA和ACTB基因荧光信号值都符合标准的“S”型曲线,熔解曲线表明所检测基因的荧光定量都具有高度的检测专一性。根据基因相对表达的计算公式:2-△△Ct ,对SAA基因表达进行相对表达量计算(常规的荧光定量PCR仪器均自带计算软件,且操作人员选择后系统可以自行计算结果)。当引物1扩增基因(SAA)的表达量相对于引物2扩增基因(ACTB)的表达量>5倍,则判断为母牛隐性子宫内膜炎为阳性;<5倍则为阴性。
最后应说明的是:以上所述仅为本发明的优选实施例而已,并不用于限制本发明,尽管参照前述实施例对本发明进行了详细的说明,对于本领域的技术人员来说,其依然可以对前述各实施例所记载的技术方案进行修改,或者对其中部分技术特征进行等同替换。凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。

Claims (2)

1.一种检测奶牛子宫内膜细胞炎性反应的荧光定量PCR试剂盒,其特征在于:所述试剂盒包括PCR反应液、TaqDNA聚合酶混合物、SYBRGreen染料和两对引物;所述引物序列为:
引物1:上游引物序列为5’-TATGACCAGGGACCAGGTAC-3’,下游引物序列为5’-CTCTGCCCTTAGGGACTCACAG-3’;
引物2:上游引物序列为5’-ATCGGCAATGAGCGGTTCC-3’,下游引物序列为5’-GTGTTGGCGTAGAGGTCCTTG-3’。
2.权利要求1中所述的两对引物在制备离体奶牛子宫内膜细胞炎性反应检测和鉴定奶牛隐性子宫内膜炎的试剂盒中的应用。
CN201410270225.9A 2014-06-18 2014-06-18 检测奶牛子宫内膜细胞炎性反应的荧光定量pcr试剂盒及其检测方法和应用 Expired - Fee Related CN104017882B (zh)

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