CN116656823A - 检测标志物及鉴别非小细胞肺癌组织学亚型的试剂盒 - Google Patents
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Abstract
本发明涉及检测标志物及鉴别非小细胞肺癌组织学亚型的试剂盒,本发明检测标志物包括检测人染色体chr20:47444720‑47444924、chr20:47444217‑47444339或chr20:47444014‑47444136一种或多种及其组合的甲基化程度。本发明通过高通量测序,以及定点甲基化荧光定量PCR方法,发现了可以区分非小细胞肺癌中的腺癌和鳞癌的甲基化基因序列。
Description
技术领域
本发明涉及疾病检测领域,特别涉及检测标志物及鉴别非小细胞肺癌组织学亚型的试剂盒。
背景技术
肺癌在我国的发病率及死亡率在我国分别位居前列及首位,肺癌组织学类型分为小细胞肺癌及非小细胞肺癌。其中非小细胞肺癌占绝大部分,约占肺癌比例的85%。而非小细胞肺癌分为鳞癌、腺癌及大细胞癌,其中鳞癌和腺癌是非小细胞肺癌的两种主要组织学亚型,占非小细胞绝大部分,而两者的生物学行为及预后有着明显差别,其临床的治疗也有较大的差别。例如靶向药物治疗对肺腺癌效果会更加佳,因而肺腺癌患者推荐行基因检测。而肺鳞癌对免疫治疗效果更好,因而推荐做PDL-1蛋白检测。目前临床上区分肺癌的组织类型,需要对确诊为肺癌的患者行手术切除获得肿瘤组织后,才能通过病理苏木精-伊红染色法(hematoxylin-eosin staining,HE)鉴别肺癌类型。若能通过肺泡灌洗液,或者活检组织中的少量癌细胞进行分子生物学方法来鉴别肺腺癌和肺鳞癌,则可以在减少患者痛苦,以及提前及早鉴别肺癌组织学类型,以精确指导肺癌患者的治疗。既有的研究表明,不同组织类型的肿瘤中存在基因甲基化的差异,例如RASSF1A基因在宫颈癌的鳞癌中发生甲基化,但在宫颈癌腺癌中不发生甲基化。
发明内容
在本发明中提供了一种检测标志物,包括检测人染色体chr20:47444720-47444924、chr20:47444217-47444339或chr20:47444014-47444136一种或多种及其组合的甲基化程度。
在一种实施方式中,本发明提供上述检测标志物在制备检测非小细胞肺癌是鳞癌或腺癌的产品中的应用。
在一种实施方式中,本发明提供引物、探针或其组合,以上述的检测标志物为扩增的目的片段。
在一种实施方式中,提供一种鉴别非小细胞肺癌组织学亚型的试剂盒,所述试剂盒用于鉴定所述非小细胞肺癌是鳞癌或腺癌,所述试剂盒用于鉴定PREX1基因启动子所在的染色体区段甲基化。
在一种实施方式中,所述试剂盒是用于鉴定人染色体chr20:47444720-47444924、chr20:47444217-47444339或/和chr20:47444014-47444136是否甲基化的试剂盒。
在一种实施方式中,所述试剂盒是用于鉴定人染色体chr20:47444734-47444813是否甲基化的试剂盒。
在一种实施方式中,所述试剂盒是用于鉴定人染色体chr20:47444257-47444317是否甲基化的试剂盒。
在一种实施方式中,所述试剂盒是用于鉴定人染色体chr20:47444720-47444924是否甲基化的q-PCR试剂盒,所述试剂盒包括上游引物序列SEQ ID NO.7:GCGCGGTTTCGGATTTTCGG,下游引物序列SEQ ID NO.8:ACGCCTCCGTCGAAAACT,探针序列SEQID NO.9:TTTCGCGGGGTTTTTTCGGAGGT。
在一种实施方式中,所述试剂盒是用于鉴定人染色体chr20:47444217-47444339是否甲基化的q-PCR试剂盒,所述试剂盒包括上游引物序列SEQ ID NO.10:GCGGCGTTTAGTTTCGGT,下游引物序列SEQ ID NO.11:CAACTAACGCTCGAACTCCC,探针序列SEQID NO.12:TTCGGTTCGTGCGCGGTC。
在一种实施方式中,所述试剂盒是用于鉴定人染色体chr20:47444014-47444136是否甲基化的q-PCR试剂盒,所述试剂盒包括上游引物序列SEQ ID NO.13:TTCGTTTCGTTGCGGTT,下游引物序列SEQ ID NO.14:CGACAACTCTAAAAAAACTCGA,探针序列SEQID NO.15:TTCCGCGCGCCCTACGA。
本发明致力于用分子生物学方法解决临床鉴别非小细胞肺癌组织学亚型的问题,本发明可以用于鉴定所述非小细胞肺癌是鳞癌或腺癌问题。在本发明中,本发明通过高通量测序,以及定点甲基化荧光定量PCR方法,发现了可以区分肺癌腺癌和鳞癌的甲基化基因序列。
附图说明
为了更清楚地说明本申请实施例中的技术方案,下面将对实施例中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本申请中记载的一些实施例,对于本领域普通技术人员来说,在不付出创造性劳动的前提下,还可以根据这些附图获得其它的附图。
图1是基于PREX1基因上的靶片段(虚线部分)和内参Actb基因(实线部分)在肺腺癌中的反应曲线示意图;
图2是基于PREX1基因上的靶片段(虚线部分)和内参Actb基因(实线部分)在肺鳞癌中的反应曲线示意图。
具体实施方式
为了使本领域技术领域人员更好地理解本申请中的技术方案,下面将结合以下实施例对本发明作进一步说明,显然,所描述的实施例仅仅是本申请一部分实施例,而不是全部的实施例。基于本申请中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其它实施例,都应当属于本申请保护的范围。
实施例一.PREX1基因启动子所在的染色体区段甲基化高通量测序
为了发现肺腺癌和肺鳞癌的甲基化差异位点,我们设计了3对甲基化引物进行PCR扩增,以亚硫酸盐转化后的基因组DNA为模板。
用以上三对引物进行甲基化PCR扩增后的产物经磁珠纯化后,使用NANODROP ONE(ThermoFisher Scientific,美国)进行浓度与纯度测定,并将纯化后的PCR产物3’端通过酶反应体系加A(腺嘌呤碱基),具体为:10×Klenow buffer 2.5μL,dATP 0.15mM,KlenowFragment(3'→5'exo-)2μL,T4 Polynucleotide kinase 2μL,总体积25μL,37℃孵育30min。3’端完成加A的PCR产物经纯化后作为模板以T4 DNA ligase 23℃孵育1h或16℃过夜连接barcode接头。连接产物经琼脂糖凝胶电泳确定目标片段在300-400bp后经纯化回收作为P5/P7通用引物扩增模板并以T100TM Thermal Cycle(BIO-RAD,美国)进行扩增(95℃预变性5min;95℃变性30sec,58℃退火30s,72℃延伸30s,17个循环)。该扩增产物经琼脂糖电泳质检后纯化富集,以Qubit 2.0Fluorometer测定浓度,通过Illumina NovaSeq测序仪(Illumina,美国)进行测序。测序结果如表1、表2和表3所示。
表格中的数字代表甲基化含量。例如第一个区段(表1)的第1个CpG岛,在第一个腺癌标本中的数字是0.412635,在第一个鳞癌样本中的数字是0。
在第一个腺癌标本中的数字是0.412635,可以这么理解,用NGS测序后,第1个CpG岛的地方,会检出CG,或者TG的序列。CG表示发生了甲基化的序列,经过亚硫酸盐转化后,CG还是CG序列。TG表示没有发生甲基化的序列,经过亚硫酸盐转化后原来基因组DNA上的CG就变成了TG;CG序列数占(CG+TG)序列数的比值为0.412635。
表1
表2
表3
肺腺癌中高度甲基化的三段区段的分析:
第一个区段,位于chr20:47444720-47444924,Human/hg19,Strand=minus
其中,chr20:47444734-47444813为优选的甲基化区段,这段区域CpG岛更加富集,适合焦磷酸测序或者甲基化荧光定量等简单方法进行分析。
第二个区段,位于chr20:47444217-47444339,Human/hg19,Strand=minus
其中,chr20:47444257-47444317为优选的甲基化区段,这段区域CpG岛更加富集,适合焦磷酸测序或者甲基化荧光定量等简单方法进行分析。
第三个区段,位于chr20:47444014-47444136,Human/hg19,Strand=minus
实施例二通过q-PCR测定不同的病例样品
在以上三段序列的方框处分别设计上游引物和下游引物,涂深色处设计探针;每条引物和探针上至少带有2个CpG岛序列,以便充分区分甲基化的CpG和非甲基化的CpG;非甲基化的CpG经过重硫酸盐处理后转化为TG序列,而甲基化的CpG序列经过重硫酸盐处理后继续保持CG序列。
三个片段设计的甲基化扩增引物和探针如下表4:
表4
以上靶片段的探针荧光修饰基团和淬灭基团都为FAM-BHQ1,内参基因Actb的引物、探针序列如下:
Actb-上游引物5’-GTGATGGAGGAGGTTTAGTAAGTT-3’(SEQ ID NO.16)
Actb-下游引物5’-CCAATAAAACCTACTCCTCCCTTAA-3’(SEQ ID NO.17)
Actb-探针5’-ACCACCACCCAACACACAATAACAAACACA-3’(SEQ ID NO.18)
Actb的探针荧光修饰基团和淬灭基团为VIC-BHQ1
经过重硫酸盐处理后,以上三段序列转化成如下序列,其中Y碱基表示或者是C碱基或者是T碱基。
第一个区段,位于chr20:47444720-47444924,Human/hg19,Strand=minus
第二个区段,位于chr20:47444217-47444339,Human/hg19,Strand=minus
第三个区段,位于chr20:47444014-47444136,Human/hg19,Strand=minus
对三段序列设计的引物探针分别和内参基因Actb的引物探针混合为一管进行双重qPCR反应。反应体系用Promega公司生产的GoTaq反应体系,具体反应体系为以下表5:
表5
qPCR反应体系 | 组成 |
5×GoTaq buffer | 4μl |
靶片段上游引物 | 0.25μM(终浓度) |
靶片段下游引物 | 0.25μM(终浓度) |
靶片段甲基化探针 | 0.40μM(终浓度) |
Actb上游引物 | 0.15μM(终浓度) |
Actb下游引物 | 0.15μM(终浓度) |
Actb探针 | 0.20μM(终浓度) |
亚硫酸盐转化后的DNA模板 | 5μl |
加入0.5μlGoTaq热启动酶,补水到20ml
反应条件:
65℃收集荧光。在内参基因的Ct<38时,实验成功;此情况下,若任一一个靶基因片段的qMS-PCR的Ct值<40,则认为甲基化检测阳性。
基于以上靶片段序列和Actb的qMS-PCR检测肺腺癌和肺鳞癌组织的代表曲线图如图1和图2。如图1所示,基于PREX1基因上的靶片段(虚线部分)和内参Actb基因(实线部分)在肺腺癌中的反应曲线,靶序列在腺癌中是高度甲基化的,故qPCR反应阳性;而在图2中,基于PREX1基因上的靶片段(虚线部分)和内参Actb基因(实线部分)在肺鳞癌中的反应曲线,靶序列在鳞癌中是去甲基化的,故qPCR反应阴性。
针对以上三个片段设计的qMS-PCR,用于51例非小细胞肺癌中的腺癌(LAC),和39例非小细胞肺癌中的鳞癌(LSC)患者的肺泡灌洗液进行测试,当3个片段中有1个片段检测的甲基化为阳性时,则判为该标本检出阳性,得到的结果如下表6,
表6
因此通过这三个片段的qMS-PCR为阳性,从肺泡灌洗液判断患者为非小细胞肺癌中的腺癌的灵敏度为94.1%(48/51),特异性为95.8%(68/71)。
应该理解到披露的本发明不仅仅限于描述的特定的方法、方案和物质,因为这些均可变化。还应理解这里所用的术语仅仅是为了描述特定的实施方式方案的目的,而不是意欲限制本发明的范围,本发明的范围仅受限于所附的权利要求。
本领域的技术人员还将认识到,或者能够确认使用不超过常规实验,在本文中所述的本发明的具体的实施方案的许多等价物。这些等价物也包含在所附的权利要求中。
Claims (10)
1.检测标志物,其特征在于,包括检测人染色体chr20:47444720-47444924、chr20:47444217-47444339或chr20:47444014-47444136一种或多种及其组合的甲基化程度。
2.根据权利要求1所述的检测标志物在制备检测非小细胞肺癌是鳞癌或腺癌的产品中的应用。
3.引物、探针或其组合,其特征在于,以如权利要求1的所述检测标志物为扩增的目的片段。
4.鉴别非小细胞肺癌组织学亚型的试剂盒,其特征在于,所述试剂盒用于鉴定所述非小细胞肺癌是鳞癌或腺癌,所述试剂盒用于鉴定PREX1基因启动子所在的染色体区段甲基化。
5.根据权利要求4中所述的试剂盒,其特征在于,所述试剂盒是用于鉴定人染色体chr20:47444720-47444924、chr20:47444217-47444339或/和chr20:47444014-47444136是否甲基化的试剂盒。
6.根据权利要求4中所述的试剂盒,其特征在于,所述试剂盒是用于鉴定人染色体chr20:47444734-47444813是否甲基化的试剂盒。
7.根据权利要求4中所述的试剂盒,其特征在于,所述试剂盒是用于鉴定人染色体chr20:47444257-47444317是否甲基化的试剂盒。
8.根据权利要求4中所述的试剂盒,其特征在于,所述试剂盒是用于鉴定人染色体chr20:47444720-47444924是否甲基化的q-PCR试剂盒,所述试剂盒包括上游引物序列SEQID NO.7:GCGCGGTTTCGGATTTTCGG,下游引物序列SEQ ID NO.8:ACGCCTCCGTCGAAAACT,探针序列SEQ ID NO.9:TTTCGCGGGGTTTTTTCGGAGGT。
9.根据权利要求4中所述的试剂盒,其特征在于,所述试剂盒是用于鉴定人染色体chr20:47444217-47444339是否甲基化的q-PCR试剂盒,所述试剂盒包括上游引物序列SEQID NO.10:GCGGCGTTTAGTTTCGGT,下游引物序列SEQ ID NO.11:CAACTAACGCTCGAACTCCC,探针序列SEQ ID NO.12:TTCGGTTCGTGCGCGGTC。
10.根据权利要求4中所述的试剂盒,其特征在于,所述试剂盒是用于鉴定人染色体chr20:47444014-47444136是否甲基化的q-PCR试剂盒,所述试剂盒包括上游引物序列SEQID NO.13:TTCGTTTCGTTGCGGTT,下游引物序列SEQ ID NO.14:CGACAACTCTAAAAAAACTCGA,探针序列SEQ ID NO.15:TTCCGCGCGCCCTACGA。
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