CN116694820A - 用于检测cmv病毒的原位杂交探针、探针组、试剂盒及检测方法 - Google Patents
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Abstract
本发明公开了一种用于检测CMV病毒的原位杂交探针、探针组、试剂盒及检测方法。其中所述用于CMV病毒检测的原位杂交探针的序列如SEQ ID No:1‑3任一所示,可用于检测CMV病毒编码的长链非编码RNA分子——lncRNA2.7。所述的探针组包括两种及两种以上不同核苷酸序列的原位杂交探针;试剂盒中包含有任一所述的探针或包含有所述的探针组,还包含有杂交液、封闭液、消化液、HRP标记的地高辛抗体,以及DAB浓缩显色液和DAB底物缓冲液。本发明公开的用于CMV病毒检测的原位杂交试剂盒具有检测速度快的优点。
Description
技术领域
本发明涉及一种用于检测CMV病毒的原位杂交探针、探针组、试剂盒及检测方法,属于基因检测技术领域。
背景技术
人巨细胞病毒(human cytomegalovirus,HCMV)属于疱疹病毒β亚科,又称人疱疹病毒5型(HHV-5),是人类疱疹病毒组中最大的一种病毒,在人群中存在普遍感染,根据不同国家和地区其感染率可达70%~100%。
HCMV感染后,可长期潜伏于骨髓单核细胞。免疫系统健康的人群,HCMV感染通常无症状,一旦免疫受损,HCMV就会被重新激活,从而导致严重的疾病或死亡。如实体器官移植术后的HCMV感染,又叫“移植的巨魔”。
近年来,随着对HCMV的研究更加深入,发现HCMV感染与肿瘤的发生发展联系密切,从而受到广泛关注。HCMV感染可以发生在多种器官,并持续感染多种成体干细胞,使之表达与潜伏期相关的HCMV蛋白,从而促进干细胞增殖、造成基因组不稳定并导致免疫逃逸。感染引起的慢性炎症可诱导活性氧的产生和先天宿主免疫防御反应,会进一步提高基因组的不稳定性,导致癌前病变(如结肠息肉)及癌变。HCMV在大脑中能感染神经元和神经胶质干细胞,维持肿瘤细胞生存、诱导致癌通路活化、提高肿瘤细胞的侵袭性等,从而增加致癌风险。
目前已知参与HCMV感染过程的病毒lncRNA主要包括lncRNA1.2、lncRNA2.7、lncRNA 4.9和lncRNA5.0,部分lncRNA的调控作用已获得证实,如lncRNA2.7可通过与线粒体酶复合物结合而抑制细胞凋亡,lncRNA5.0与HCMV的毒力有密切关系。lncRNA4.9直到最近才从HCMV表达谱中被确认,其转录本长度为4922bp。其中,lncRNA 2.7是CMV病毒增殖感染阶段丰度最高的早期基因转录产物。因此,相比与CMV病毒表达的其他RNA分子,lncRNA2.7是理想的CMV病毒检测标志物。但采用当前免疫组化手段检测CMV病毒时,检测到病毒的时间存在一定程度上的滞后;且现有检测方法的检测时间较长。
发明内容
本发明的目的在于提供一种用于检测CMV病毒的原位杂交探针、探针组、试剂盒及检测方法,其能够检测CMV编码的长链非编码RNA分子——lncRNA 2.7,可以增加CMV的检出率,为CMV患者的早期诊断提供有价值的参考信息。
本发明所采用的技术方案为:
用于检测CMV病毒的原位杂交探针,所述探针的核苷酸序列如SEQ ID NO:1~SEQID NO:3中任意一种所示。
优选地,所述探针的其中一个末端修饰有可检测标记物;所述的可检测标记物为地高辛、生物素、罗丹明中的任意一种。
优选地,所述探针的另一个末端也修饰有可检测标记物;所述的可检测标记物为地高辛、生物素、罗丹明中的任意一种。
用于检测CMV病毒的探针组,包括两种及两种以上不同核苷酸序列的上述原位杂交探针。
用于检测CMV病毒的试剂盒,其中包含有任意上述的探针或包含有上述的探针组。
优选地,还包括有封闭液、消化液、HRP标记的地高辛抗体,以及DAB浓缩显色液和DAB底物缓冲液。
优选地,还包括杂交液,所述杂交液由6×SSC缓冲液、5×Denhardt溶液、SDS溶液与Salmon sperm DNA溶液混合而成。
用于检测CMV病毒的检测方法,该方法使用任意上述的探针或使用上述的探针组或使用任意上述的试剂盒进行检测。
优选地,所述方法依次包括脱蜡、封闭、消化、杂交、洗涤、结合、显色、复染-脱水-封片、阅片步骤。
优选地,杂交条件为65℃-100℃变形3-10min后,于30℃-45℃杂交1-2.5小时。
本发明的有益效果在于:
本发明提供的探针、探针组以及试剂盒能够检测CMV编码的长链非编码RNA分子——lncRNA 2.7,并可以增加CMV的检出率,能够为CMV患者的早期诊断提供有价值的参考信息;在检测时,通过调整CMV探针孵育温度,将检测时间由过夜(8-12小时)缩短至2小时,大幅提高了杂交效率。
附图说明
图1为lncRNA 2.7在细胞中的结构图;
图2为探针检测效果图,其中A-C分别为SEQ ID NO:1-NO:3的检测效果图;
图3为CMV病毒原位杂交检测与免疫组化检测对比图;其中,A区域为SEQ ID NO:1的探针原位杂交检测的效果图;B区域为免疫组化检测的效果图;
图4为不同杂交条件对比图。A为37℃过夜杂交;B为37℃杂交4小时;C为37℃杂交2小时。
具体实施方式
下面结合附图和实施例对本发明做具体的介绍。
用于CMV检测的原位杂交探针的筛选
为验证原位杂交探针的设计质量和确定最佳的结合区域,本发明针对CMV编码的lncRNA 2.7的序列,分别设计了3组DNA探针进行平行比较,其序列如表1所示。
表1原位杂交探针序列
待测靶标lncRNA 2.7的序列和结果则分别如表2和图1所示。
表2lncRNA2.7的序列
本实施例中用于CMV病毒检测的原位杂交试剂盒及其使用方法如下:
1.试剂准备
1.1委托基因合成公司合成表1所述序列,并在其5’和3’端均修饰上Digoxin;
1.2将合成好的探针用纯化水溶解,并使用杂交液(6×SSC;5×Denhardt;0.5%SDS;100μg/ml Salmon sperm DNA)将其浓度稀释到1~2ng/μL;
1.3按19:1的比例分别加入DAB底物缓冲液和DAB浓缩显色液,将其均匀混合,配制成DAB工作液。
2.操作流程
2.1脱蜡:将新鲜的石蜡切片放入二甲苯依次脱蜡3x10min,去除多余液体后,放入无水乙醇中3x3min,然后空气干燥5-10min;
2.2封闭:向干燥后的切片上滴加80-100μL的5%过氧化氢封闭液,室温避光孵育10min后用纯水进行洗涤,然后用75%、95%、100%乙醇溶液梯度脱水各2min,空气晾干;
2.3消化:向干燥后的切片上滴加80-100μL的100μg/mL胃蛋白酶消化液,37℃孵育5-30min后用纯水进行洗涤,然后用75%、95%、100%乙醇溶液梯度脱水各2min,空气晾干;
2.4杂交:向干燥后的切片上滴加5-10μL的1~2ng/μL探针杂交液,盖上硅化盖玻片,橡胶水泥封边,95℃变性5分钟后于37℃杂交孵育过夜;
2.5洗涤:小心去除橡胶水泥,将切片放入PBS缓冲液中揭下盖玻片,然后浸泡清洗10min;
2.6结合:向切片上滴加30-50μL的HRP标记的Digoxin抗体,37℃孵育30min后放入PBS缓冲液中清洗10min;
2.7显色:向切片上滴加80-100μL的新配制的DAB工作液,室温孵育5-10min;
2.8复染-脱水-封片:用纯化水清洗掉多余的DAB工作液,然后滴加10-30μL的苏木素染色液,室温孵育10-30s后用75%、95%、100%乙醇溶液梯度脱水各2min,空气晾干,最后用中性树胶封片;
2.9阅片:显微镜下观察探针杂交情况,阳性染色定位于细胞浆。
SEQ ID NO:1-NO:3对应探针的杂交情况如图2所示,结果显示,SEQ ID NO:1对应探针的检测结果符合浆阳性的特征,优于SEQ ID NO:2和SEQ ID NO:3对应的探针;也即,SEQ ID NO:1对应的探针在检测效果上优于SEQ ID NO:2和NO:3。
CMV病毒原位杂交检测与免疫组化检测对比
CMV病毒原位杂交检测与免疫组化检测的对比图如图3所示。其中图3A区域为SEQID NO:1的探针原位杂交检测的效果图;图3B区域为免疫组化检测的效果图。结果显示针对CMV病毒感染早期表达的lncRNA 2.7原位杂交检测的信号强度不弱于免疫组化检测CMV病毒感染晚期表达的蛋白的信号强度,表明CMV原位杂交检测能够有效增加CMV病毒感染的早期检出率。
也即本发明SEQ ID NO:1对应的探针针对CMV病毒感染早期表达的lncRNA2.7所设计,相比免疫组化检测CMV病毒表达的蛋白,能够更早提示CMV病毒的侵入,有效增加CMV病毒早期感染的检出率。
CMV探针杂交温度及时间优化
如图1所示,CMV病毒表达的lncRNA 2.7具有相当复杂的二级结构,相比专利CN114182048A的探针杂交温度及时间(70℃变性8分钟后37℃过夜杂交),本发明将探针杂交温度和时间变更为95℃变性5分钟后37℃分别杂交4和2小时,结果如图4所示,3种不同条件下的探针杂交效果无明显变化,表明95℃变性5分钟,充分打开了lncRNA 2.7的二级结果,提高了CMV探针的结合效率,大幅减少杂交时间。
综上所述,本发明公开的用于检测CMV病毒的原位杂交探针、探针组、试剂盒及检测方法,能够检测CMV编码的长链非编码RNA分子——lncRNA 2.7,可以增加CMV的检出率,为CMV患者的早期诊断提供有价值的参考信息。
以上所述仅是本发明专利的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明专利原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明专利的保护范围。
Claims (10)
1.用于检测CMV病毒的原位杂交探针,其特征在于,所述探针的核苷酸序列如SEQ IDNO:1~SEQ ID NO:3中任意一种所示。
2.根据权利要求1所述的用于检测CMV病毒的原位杂交探针,其特征在于,所述探针的其中一个末端修饰有可检测标记物;所述的可检测标记物为地高辛、生物素、罗丹明中的任意一种。
3.根据权利要求2所述的用于检测CMV病毒的原位杂交探针,其特征在于,所述探针的另一个末端也修饰有可检测标记物;所述的可检测标记物为地高辛、生物素、罗丹明中的任意一种。
4.用于检测CMV病毒的探针组,其特征在于,包括两种及两种以上不同核苷酸序列的如权利要求1-3中任意一项所述的原位杂交探针。
5.用于检测CMV病毒的试剂盒,其特征在于,其中包含有权利要求1-3中任一所述的探针或包含有权利要求4中所述的探针组。
6.根据权利要求5所述的用于检测CMV病毒的试剂盒,其特征在于,还包括有封闭液、消化液、HRP标记的地高辛抗体,以及DAB浓缩显色液和DAB底物缓冲液。
7.根据权利要求6所述的用于检测CMV病毒的试剂盒,其特征在于,还包括杂交液,所述杂交液由6×SSC缓冲液、5×Denhardt溶液、SDS溶液与Salmon sperm DNA溶液混合而成。
8.用于检测CMV病毒的检测方法,其特征在于,使用权利要求1-3中任一所述的探针或使用权利要求4中所述的探针组或使用权利要求5-7中任一所述的试剂盒进行检测。
9.根据权利要求8所述的用于检测CMV病毒的检测方法,其特征在于,依次包括脱蜡、封闭、消化、杂交、洗涤、结合、显色、复染-脱水-封片、阅片步骤。
10.根据权利要求9所述的用于检测CMV病毒的检测方法,其特征在于,杂交条件为85℃-100℃变形3-10min后,于30℃-45℃杂交1-2.5小时。
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