CN105543411B - 检测IFFO1基因mRNA的可变腺苷酸位点使用情况的引物及方法 - Google Patents
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Abstract
本发明公开了检测IFFO1基因mRNA的可变腺苷酸位点使用情况的引物及方法。所述引物包括第一对引物和第二对引物,其序列与IFFO1基因3’非翻译区中的两对序列或其互补序列相同。所述方法包括以下步骤:提取样品总RNA,逆转录得到cDNA;以cDNA为模板,使用第一对引物qPCR扩增common区域的Proximal片段,得到CT值pro;以cDNA为模板,使用第二对引物扩增Extended区域的Distal片段,得到CT值dis;计算CT值dis与CT值pro的比值。本发明IFFO1基因mRNA的可变腺苷酸位点使用情况的检测方法方便快捷,适合在条件简陋的实验室以及发展中国家得到进一步推广。
Description
技术领域
本发明涉及病毒检测技术领域,尤其涉及检测IFFO1基因mRNA的可变腺苷酸位点使用情况的引物及方法。
背景技术
马立克氏病(Marek's disease,MD)是由马立克氏病病毒((Marek's diseasevirus,MDV)引起的一种禽类高度接触性、传染性、淋巴组织增生性肿瘤病,主要危害鸡,鹤鹑、山鸡也有发病。该病可引起内脏器官、肌肉和外周神经的淋巴细胞瘤的神经肿大。临床上MD发病鸡群表现为生长不均匀、极度消瘦、死亡等症状,最常见的病变是神经损害和多种实质脏器的淋巴肿瘤,以肝脏、脾脏肿瘤最为多见,因神经损害导致的不对称性进行性麻痹,最终可引起单个或多个肢体的完全性麻痹。
MDV属于疱疹病毒科,有三种不同血清型,不同血清型毒株既含有共同抗原,也含有特异性抗原。Ⅰ型MDV感染能引起肿瘤,Ⅱ型和Ⅲ型MDV毒株无致病性,可用于疫苗株。因Ⅰ型MDV感染可引起肿瘤,其检测具有非常重要的意义。目前,临床上最常用鉴别诊断Ⅰ型MDV感染的方法是病毒分离培养、血清学检测、分子生物学方法等。病毒分离鉴定不但对临床诊断很有价值,对流行病学及病原学研究亦至关重要,但该方法检测周期长,约需1~2个月,同时细胞培养的操作要求高,局限性较大。血清学方法如ELISA等主要用于检测羽毛囊上皮、溶细胞感染的淋巴细胞组织及细胞培养物等样品,此类方法检测成本相对较高,并且由于部分病例中病毒血症时间较短,病毒含量低,导致检出率较低。
发明内容
有鉴于此,有必要针对现有技术中检测马立克氏病病毒存在的问题,提供IFFO1(intermediate filament family orphan 1,IFFO1)基因mRNA的可变腺苷酸位点使用情况的检测引物及检测方法。
为了实现上述目的,本发明采用如下技术方案:
一种检测IFFO1基因mRNA的可变腺苷酸位点使用情况的引物,所述引物包括第一对引物和第二对引物,其序列与IFFO1基因3’非翻译区中的两对序列或其互补序列相同。
优选地,所述第一对引物的序列与IFFO1基因3’非翻译区中common区域的部分序列或其互补序列相同,所述第二对引物的序列与IFFO1基因3’非翻译区中Extended区域的部分序列或其互补序列相同。
优选地,所述第一对引物用于扩增common区域的Proximal片段,所述第二对引物用于扩增Extended区域的Distal片段。
优选地,所述第一对引物的序列如SEQ ID NO:1-2所示或其互补序列,所述第二对引物的序列如SEQ ID NO:3-4所示或其互补序列。
一种检测IFFO1基因mRNA的可变腺苷酸位点使用情况的方法,包括以下步骤:
提取样品总RNA,逆转录得到cDNA;
以cDNA为模板,使用第一对引物qPCR扩增common区域的Proximal片段,得到CT值pro;
以cDNA为模板,使用第二对引物扩增Extended区域的Distal片段,得到CT值dis;
计算CT值dis与CT值pro的比值。
优选地,所述qPCR的反应体系为:
优选地,所述qPCR的反应条件为:
预变性:95℃1min;扩增:95℃5s,62℃15s,72℃10s信号收集,40个循环;溶解曲线:95℃1min;冷却:37℃1min。
3’端非翻译区(3’UTR)作为mRNA结构的一部分,对mRNA的稳定性、定位及翻译效率都起着重要的作用。基因最后一个外显子上的可选择性poly(A)位点,可以导致不同长度的3’UTR,即tandem 3’UTR(串联3’UTR)。目前已有大量文献报道在免疫应答、神经活化、肿瘤形成及胚胎发育过程中均有3’UTR长度的改变。在基因动态的调节过程中,选择性聚腺苷酸化(alternative polyadenylation,APA)在近年来受到人们的广泛关注。最近的研究表明有超过一半的人类基因含有APA位点。这些都暗示着APA对于基因功能可能有着重要的调控作用,也有着巨大的潜力成为新的分子标记物。随着第二代高通量测序技术的发展,研究人员近年来开发了多种高通量测序文库制备方法来对全基因组APA进行测序和分析。随着APA研究的深入,APA越发成为新的基因转录与翻译调控的研究热点,目前,已有研究开始将APA作为肿瘤发生的生物学标记。
IFFO1基因是中间丝家族的成员,中间丝蛋白则是细胞骨架和核膜的原始组成部分。本发明利用鸡的IFFO1基因3’非翻译区中的两对序列或其互补序列,通过实时荧光定量PCR方法对鸡只组织或血液中的IFFO1基因信使核糖核酸mRNA的可变腺苷酸位点使用情况进行检测,发现宿主在感染MDV后IFFO1基因可变腺苷酸位点的使用情况发生了明显改变,可将IFFO1基因可变腺苷酸位点的使用变化作为宿主(鸡只等)感染马立克病毒的生物学标记。
与现有技术相比,本发明具有如下有益效果:本发明IFFO1基因mRNA的可变腺苷酸位点使用情况的检测方法方便快捷,灵敏度高,特异性好,适合在条件简陋的实验室以及发展中国家得到进一步推广。
附图说明
图1为mRNA结构中qPCR引物设计位点示意图。
图2为实施例1中IFFO1基因APA的变化情况。
具体实施方式
为了更好的说明本发明,下面结合附图和具体实施方式做进一步说明。本发明中所用试剂或仪器均可由市场购得,使用的检测方法等都是本领域所熟知的,在此不再赘述。
实施例1
用1×105pfu Md5(Marek's disease virus Md5,MDV Md5)感染单层CEF细胞(鸡胚成纤维细胞),以不感染的CEF细胞为对照,分别收集18h、36h、54h、72h后的细胞,提取总RNA;消化DNA后,检测OD260/280,比值在2.0-2.1为合格。对合格的总RNA进行SAPAS(sequence of alternative polyadenylation site,SAPAS)文库的构建,构建好的文库用Hi-Seq-2500平台进行高通量测序,对测序结果进行APA(alternative polyadenylation,APA)的分析。
结果如图2所示,对比感染Md5和不感染Md5的CEF细胞的APA分析结果,发现IFFO1基因多聚腺苷酸化位点的使用情况发生了明显改变,感染Md5的CEF细胞的IFFO1基因更偏向于使用近端的APA位点,可作为感染MDV的一个生物标记。
实施例2
本实施例中检测IFFO1基因mRNA的可变腺苷酸位点使用情况的引物如表1所示。该引物是根据IFFO1基因(gene bank Accession:XM_416501)用primer premier 5设计得到的。第一对引物根据common区域进行设计,用于扩增common区域的Proximal片段;第二对引物根据Extended区域进行设计,用于扩增Extended区域的Distal片段。两对引物由invitrogen公司合成。
表1、引物序列表
检测IFFO1基因mRNA的可变腺苷酸位点使用情况的方法,包括以下步骤:
S1、提取样品总RNA,逆转录得到cDNA
用1×105pfu Md5(Marek's disease virus Md5,MDV Md5)感染单层CEF细胞(鸡胚成纤维细胞),以不感染的CEF细胞为对照,分别收集18h、36h、54h、72h后的细胞,提取总RNA;消化DNA后,检测OD260/280,比值在2.0-2.1为合格。采用常规方法对合格的总RNA进行逆转录,得到cDNA。
S2、qPCR
使用第一对引物以cDNA为模板qPCR扩增common区域的Proximal片段,其CT值记为CT值pro;以cDNA为模板,使用第二对引物扩增Extended区域的Distal片段,其CT值记为CT值dis;计算CT值dis与CT值pro的比值。
其中,qPCR反应体系如下:
qPCR反应条件如下:
预变性:95℃1min;扩增:95℃5s,62℃15s,72℃10s信号收集,40个循环;溶解曲线:95℃1min;冷却:37℃1min。
如图1所示,用IFFO1-short-F、IFFO1-short-R这对引物扩增的proximal片段反映的是较为近端的3’UTR isoform的表达情况,proximal片段扩增的量可以反映总的3’UTRisoform的量;而相应的用IFFO1-long-F、IFFO1-long-R扩增的distal扩增片段是远端的3’UTR isoform的表达情况,distal片段扩增的量可以反映长的3’UTR isoform的表达量。因此,CT值dis/CT值pro可以反映样品中该基因3’UTR isoform的使用情况:CT值dis/CT值pro越小则说明样品中使用短的3’UTR isoform越多,相应地,CT值dis/CT值pro越大则说明样品中使用长的3’UTR isoform越多。因此,通过qPCR的CT值比较可获得IFFO1的可变腺苷酸位点的使用情况。
通过计算可知,对照组的CT值dis/CT值pro平均值为0.37,感染病毒组的CT值dis/CT值pro平均值降为0.23。相对于对照组,感染病毒组可变腺苷酸位点的使用明显偏向使用近端的3’UTR,与实施例1的结果相符。
实施例3
以2000pfu Md5感染一日龄小鸡十只,30天后采集感染Md5鸡只全血,同时采集十只未感染MDV的鸡只全血,分别从血液中分离淋巴细胞作为样本,进行IFFO1基因mRNA的可变腺苷酸位点使用情况检测,检测方法与实施例2相同。本实施例中对照组的CT值dis/CT值pro平均值为0.41,感染病毒组的CT值dis/CT值pro平均值降为0.22,结果与实施例2一致,表明IFFO1基因多聚腺苷酸化位点的使用情况可作为鸡感染MDV的一个生物标记。
以上所述实施例仅表达了本发明的几种实施方式,其描述较为具体和详细,但并不能因此而理解为对本发明专利范围的限制。应当指出的是,对于本领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干变形和改进,这些都属于本发明的保护范围。因此,本发明专利的保护范围应以所附权利要求为准。
Claims (5)
1.一种检测IFFO1基因mRNA的可变腺苷酸位点使用情况的引物,其特征在于,所述引物包括第一对引物和第二对引物,其序列与IFFO1基因3’非翻译区中的两对序列或其互补序列相同;
所述第一对引物的序列与IFFO1基因3’非翻译区中common区域的部分序列或其互补序列相同,所述第二对引物的序列与IFFO1基因3’非翻译区中Extended区域的部分序列或其互补序列相同;
所述第一对引物用于扩增common区域的Proximal片段,所述第二对引物用于扩增Extended区域的Distal片段;
所述第一对引物的序列如SEQ ID NO:1-2所示或其互补序列,所述第二对引物的序列如SEQ ID NO:3-4所示或其互补序列。
2.一种检测IFFO1基因mRNA的可变腺苷酸位点使用情况的非诊断方法,其特征在于,使用权利要求1所述的引物,包括以下步骤:
提取样品总RNA,逆转录得到cDNA;
以cDNA为模板,使用第一对引物qPCR扩增common区域的Proximal片段,得到CT值pro;
以cDNA为模板,使用第二对引物扩增Extended区域的Distal片段,得到CT值dis;
计算CT值dis与CT值pro的比值。
3.根据权利要求2所述的方法,其特征在于,所述第一对引物的序列如SEQ ID NO:1-2所示或其互补序列,所述第二对引物的序列如SEQ ID NO:3-4所示或其互补序列。
5.根据权利要求2所述的方法,其特征在于,所述qPCR的反应条件为:预变性:95℃1min;扩增:95℃5s,62℃15s,72℃10s信号收集,40个循环;溶解曲线:95℃1min;冷却:37℃1min。
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