CN111235266A - Hla亚型检测试剂盒及其应用 - Google Patents
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Abstract
本发明涉及HLA亚型检测试剂盒,所述试剂盒包括用于检测多种HLA亚型的多重PCR扩增的引物和探针,每对HLA亚型上游和下游引物的量比值为1:20到1:50,每对引物上游引物的Tm值比下游引物的Tm值高4‑8℃。本发明结合通过提高限速引物的退火温度,加上调整上下游引物的配比,能较传统制备单链方法在不损失PCR扩增效率的情况下,制备出更多的单链,从而使得探针的杂交信号较传统的多重单链制备PCR得到明显的提升,从而提高了检测的灵敏度和特异性。
Description
技术领域
本发明涉及分子诊断领域,特别涉及HLA亚型检测试剂盒及其应用。
背景技术
癫痫是神经科第二常见疾病,我国癫痫患者过千万,每年新增约40万,药物治疗为癫痫的主要治疗方式,多数患者需要长期服药,但大约16%的患者出现皮肤型过敏反应,包括轻度的斑丘疹(maculopapular exanthema,MPE)及重度的斯蒂文森-强生综合症(Stevens-Johnson syndrome,SJS)和中毒性表皮坏死松解症(toxic epidermalnecrolysis,TEN),明显影响生活质量,严重时出现剥脱性皮炎,通常全身粘膜(眼、嘴、生殖器)溃疡,皮肤起水疱,甚至出现全身表皮脱离,如同大面积烫伤,致死性高达40%。这种过敏在南方汉族人群,尤其是在广东及周边地区的汉族人群中发病率明显高于其它地区及人群。
卡马西平(CBZ)、拉莫三嗪(LTG)、奥卡西平(OXC)、苯妥英钠(PHT)以及妥泰(PB)都具有相似的芳香族结构,是广泛使用的用于控制癫痫、三叉神经痛等的抗癫痫药物(AEDs)。然而,该类药物通常会导致皮肤不良反应(cADRs),长期困扰着临床医生。cADRs占所有报道的药物不良反应的10%-30%,包括轻度的斑丘疹(MPE)、药物超敏反应综合症(HSS)到重型的有潜在致命性的Stevens-Johnson综合症(SJS)和/或中毒性表皮坏死松解症(Toxicepidermal necrolysis,TEN)。如能找到与其相关的基因标志,将对临床用药起到很大的指导作用。人类白细胞抗原(HLA)基因与人体免疫相关,包括数千个不同的基因型。HLA-B*15:02为我国南方汉族人群卡马西平导致剥脱性皮炎的遗传标记。HLA-A*24:02是卡马西平导致剥脱性皮炎的风险基因,也是其它抗癫痫药物导致剥脱性皮炎的通用风险基因,除了以上两个,并且还存在其它HLA风险基因,包括HLA-A*02:01、HLA-B*15:01、HLA-B*13:01、HLA-B*15:01、HLA-B*15:11、HLA-B*35:01、HLA-B*38:02和DRB1*04:06,这些基因型可以解释约90%的癫痫患者用药过敏风险。因此癫痫患者可通过基因筛查降低甚至避免致死性皮肤型不良反应的发生,例如由于HLA-B*15:02明确为卡马西平导致剥脱性皮炎的基因标记,2008年美国FDA已建议个体服用卡马西平前进行HLA-B*15:02基因检测,以避免剥脱性皮炎的发生。而HLA-A*24:02的发现,将卡马西平导致剥脱性皮炎的排除概率从69.6%提高到82%;这些癫痫药物的主要基因标记,将多个药物导致剥脱性皮炎的排除概率达到90%,而且为其他人种及其它类型的皮肤型过敏提供通用遗传标记,惠及的个体更多。个体特别是南方汉族人,服用芳香族抗癫痫药物之前,检测主要遗传标记,医生对携带主要遗传标记的阳性个体慎用芳香族类抗癫痫药物,降低甚至避免致死性皮肤型不良反应的发生。
发明内容
为了解决上述问题,本发明提供一种HLA亚型检测试剂盒,所述试剂盒包括用于检测多种HLA亚型的多重PCR扩增的引物和探针,每对HLA亚型的上游和下游引物的量比值为1:20到1:50,每对引物的上游引物的Tm值比下游引物的Tm值高4-8℃。
在一种实施方式中,每对HLA亚型的上游和下游引物的量比值为1:30到1:40。
在一种实施方式中,在所述上游引物5’端增加碱基GCG以提高所述上游引物的Tm值。
在一种实施方式中,所述试剂盒是用于检测以下HLA亚型的试剂盒:HLA-A*02:01、HLA-A*24:02、HLA-B*15:01、HLA-B*15:02、HLA-B*15:11、HLA-B*35:01、HLA-B*38:02、HLA-B*13:01和HLA-DRB1*04:06。
在一种实施方式中,所述试剂盒包括以下引物:
在一种实施方式中,所述试剂盒包括以下探针:
检测的位点 | 探针序列(5’--3’) | 序列名称 |
HLA-A*02:01 | ACTTGTGCTTGGTGGTCTGAGCT | SEQ ID NO:19 |
HLA-A*24:02 | AAAGTGAAGGCCCACTCACAGACT | SEQ ID NO:20 |
HLA-B*15:01 | CGTCGCAGCCGTACATCCTCTGG | SEQ ID NO:21 |
HLA-B*15:02 | GACCCGCCCAAACCCTCGACC | SEQ ID NO:22 |
HLA-B*15:11 | TGGGACCGGAACACACAGAT | SEQ ID NO:23 |
HLA-B*35:01 | CGACCTGGGGCCCGACG | SEQ ID NO:24 |
HLA-B*38:02 | CTCTCGGTAAGTCTGTGTGTTGGT | SEQ ID NO:25 |
HLA-B*13:01 | CCAGGTCGCAGCCATACATCCT | SEQ ID NO:26 |
HLA-DRB1*04:06 | CGGGCCGAGGTGGACAC | SEQ ID NO:27 |
在一种实施方式中,本发明提供上述试剂盒在癫痫用药过敏预警中的应用。
由于本发明的多重PCR扩增体系中,把上游引物设置为限速引物,额外增加了碱基提高Tm值,使得每对的上游引物比下游引物高4-8℃,并使得限速引物的浓度只有下游引物的1:20到1:50,因此在PCR扩增中,能获得较多的下游引物制备的单链。传统的PCR制备单链的方法,一般就调整上游和下游引物的配比,只有当占比少的引物用完之后,才能在多余的引物上通过单引物扩增获得一定量的单链。本发明结合通过提高限速引物的退火温度,加上调整上下游引物的配比,能较传统制备单链方法在不损失PCR扩增效率的情况下,制备出更多的单链,从而使得探针的杂交信号较传统的多重单链制备PCR(每对引物仅调节上游:下游引物=1:20到1:50)得到明显的提升,从而提高了检测的灵敏度和特异性。
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是本发明和传统多重单链制备PCR方法检测9种HLA基因亚型的探针杂交信号比较图;
图2是本发明和当限速引物的Tm值较另一组引物不超过4℃时微珠上的探针杂交信号比较图;和
图3是本发明和当限速引物的Tm值较另一组引物超过8℃时微珠上的探针杂交信号比较图。
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.本发明的多重扩增9种HLA亚型检测试剂盒
1.检测9种HLA亚型引物和探针的设计
本试剂盒针对人类基因组DNA中发现的和癫痫用药过敏相关的9个HLA基因亚型:HLA-A*02:01、HLA-A*24:02、HLA-B*15:01、HLA-B*15:02、HLA-B*15:11、HLA-B*35:01、HLA-B*38:02、HLA-B*13:01和HLA-DRB1*04:06。设计特异性引物和探针,其中特异性引物进行多重复合扩增获得9个HLA基因亚型的扩增产物;探针用于偶联在悬浮微珠液相芯片的微珠上,根据最终探针的杂交信号,可以对各个HLA基因亚型进行判定。
本发明中涉及的引物、探针序列如下表1:
表1.多重扩增9种HLA亚型的PCR引物序列
其中下游引物上5’端都带有Biotin修饰。在上游引物增加了GCG三个碱基,以提高上游引物的退火温度。在PCR扩增反应体系中,上游引物的浓度:下游引物的浓度为1:40,这样就在较高的PCR反应退火温度情况下,制备出下游引物扩增的单链DNA。
和以上癫痫用药过敏相关的9个HLA基因亚型对应的探针序列如下表2。
表2.悬浮微珠液相芯片检测9种HLA基因亚型的探针序列
检测的位点 | 探针序列(5’--3’) | 序列名称 |
HLA-A*02:01 | ACTTGTGCTTGGTGGTCTGAGCT | SEQ ID NO:19 |
HLA-A*24:02 | AAAGTGAAGGCCCACTCACAGACT | SEQ ID NO:20 |
HLA-B*15:01 | CGTCGCAGCCGTACATCCTCTGG | SEQ ID NO:21 |
HLA-B*15:02 | GACCCGCCCAAACCCTCGACC | SEQ ID NO:22 |
HLA-B*15:11 | TGGGACCGGAACACACAGAT | SEQ ID NO:23 |
HLA-B*35:01 | CGACCTGGGGCCCGACG | SEQ ID NO:24 |
HLA-B*38:02 | CTCTCGGTAAGTCTGTGTGTTGGT | SEQ ID NO:25 |
HLA-B*13:01 | CCAGGTCGCAGCCATACATCCT | SEQ ID NO:26 |
HLA-DRB1*04:06 | CGGGCCGAGGTGGACAC | SEQ ID NO:27 |
以上探针在5’端都带有5’-Amino C6+Spacer 18氨基修饰。
2.试剂盒的实施过程
2.1.悬浮微珠液相芯片制备
表面修饰有羧基的磁性微珠从美国Luminex公司购得,按照美国Luminex公司提供的核酸探针偶联磁珠方案,把探针偶联到磁珠上。每条探针偶联一种编码的磁珠,9条探针一共偶联9种编码磁珠。具体过程如下:取出40μl(1×107个)个磁性微球,用磁棒吸附磁性微球后,吸去上清,加入5μl 0.1M MES(pH4.5),混匀。将所用的探针稀释至0.1mM,在偶联体系中加入1μl。第一次加入2.5μl 10mg/ml EDC,混匀后避光放置30min。重复再加入一次EDC,混匀避光放置30min。用0.2ml 0.02%Tween和0.1%SDS各洗一次,最后将微球重新悬浮于10μl TE(pH 8.0)溶液中,混匀后使用血细胞计数器计数微球数量(计数四角4个大方格的数目后换算为每微升微球个数)。4℃避光保存。混合偶联好的探针的微球,使用1.5×TMAC稀释至每种微球的浓度是150个/μl。
2.2.PCR扩增
以上9个HLA基因位点的9对PCR引物,在一管种实现多重PCR扩增,反应的体系为总体积25μl,包含浓度为5ng/μl的DNA模板5μl、10ХPCR反应缓冲液(含Mg2+)5μl、5MBetain 4μl、酶系3μl(包括25mM dNTP混合液0.9μl,10u/μl热启动Taq酶0.6μl、1μ/lUDG酶0.1μl、热启动酶稀释液1.4μl);管中引物包括终浓度为2μM的上游引物,和终浓度为80μM的下游引物(上游引物:下游引物浓度比为1:40),补足去离子水至25μL。
在BioRad公司的T-100PCR扩增仪上进行反应,反应条件为50℃、3分钟,95℃、1分钟,进行45个循环的94℃、30秒,60℃、30秒,72℃、30秒;72℃、7分钟,4℃维持。
2.3.上机检测和结果分析
取5μl PCR产物和45μl的偶联微珠,反应总体积50μL,密封后,按下列条件避光进行杂交反应:95℃变性5min,然后60℃杂交15min。然后进行显色反应。吸取显色工作液,加入杂交反应液中,每反应25μL,吹打混匀,60℃避光孵育7min。杂交产物上机检测,检测仪器为Luminex液相芯片检测仪MAGPIX。若探针的荧光信号强度都高于背景荧光信号强度3倍以上,则该探针检出为阳性。
用本发明中的单管多重PCR扩增体系,对108个经过测序已知HLA基因型样本进行检测,检测结果如下,从表3中可以看出,每种HLA亚型基因型检测的灵敏度和特异度均较高。
表3.本发明检测9种HLA基因亚型的准确性数据
HLA基因亚型 | 灵敏度 | 特异度 |
HLA-A*02:01 | 100 | 98.15 |
HLA-A*24:02 | 100 | 97.22 |
HLA-B*15:01 | 100 | 98.15 |
HLA-B*15:02 | 100 | 99.07 |
HLA-B*15:11 | 100 | 96.30 |
HLA-B*35:01 | 100 | 97.22 |
HLA-B*38:02 | 100 | 97.22 |
HLA-B*13:01 | 100 | 98.15 |
HLA-DRB1*04:06 | 100 | 97.22 |
实施例2不同条件下比较试验
由于本发明的多重PCR扩增体系中,把上游引物设置为限速引物,额外增加了碱基提高Tm值,上游引物比下游引物Tm值高4-8℃;并使得限速引物的浓度只有下游引物的1/40,因此在PCR扩增中,能获得较多的下游引物制备的单链。传统的PCR制备单链的方法,一般就调整上游和下游引物的配比,只有当占比少的引物用完之后,才能在多余的引物上通过单引物扩增获得一定量的单链。本发明结合通过提高限速引物的退火温度,加上调整上下游引物的配比,能较传统制备单链方法在不损失PCR扩增效率的情况下,制备出更多的单链,从而使得探针的杂交信号较传统的多重单链制备PCR(每对引物仅调节上游:下游引物=1:40)得到明显的提升,从而提高了检测的灵敏度和特异性,具体结果如如图1。
在本发明中,限速引物的Tm值调整为高于另一引物4-8℃。我们发现当限速引物的Tm值较另一组引物不超过4℃时,单链制备的量并不明显,从而微珠上的探针杂交信号较本发明确定的高4-8℃效果要差,具体见图2;而当限速引物的Tm值较另一引物超过8℃时,又影响了PCR的扩增效率,也导致微珠上的探针杂交信号较本发明确定的高4-8℃效果要差,具体见图3。
另外,在保持每对上游引物比下游引物高4-8℃情况下,用上述同样的方法验证了上游引物和下游引物之间比例关系对于检测的灵敏度和特异性影响,在上游引物与下游引物的量在1:20-1:50时都可以实现高灵敏度和高特异性,但是在上游引物与下游引物的量大于1:20和小于1:50时试剂盒的灵敏度和特异性有明显降低,在上游引物与下游引物的量1:30-1:40时效果最佳。
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。
序列表
<110> 广州医科大学附属第二医院
<120> HLA亚型检测试剂盒及其应用
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<170> SIPOSequenceListing 1.0
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<213> 人工序列(Artificial Sequence)
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ggctctcaac tgctccgcta caa 23
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<212> DNA
<213> 人工序列(Artificial Sequence)
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<212> DNA
<213> 人工序列(Artificial Sequence)
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gagcgcgatc cgcagatt 18
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<212> DNA
<213> 人工序列(Artificial Sequence)
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<212> DNA
<213> 人工序列(Artificial Sequence)
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<213> 人工序列(Artificial Sequence)
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<212> DNA
<213> 人工序列(Artificial Sequence)
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<212> DNA
<213> 人工序列(Artificial Sequence)
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gcgggaatat tgggaccgga ac 22
<210> 14
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<212> DNA
<213> 人工序列(Artificial Sequence)
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ttgtaatagc ggagcgtggt g 21
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<212> DNA
<213> 人工序列(Artificial Sequence)
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gcgggccagg gtctcacatc a 21
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<212> DNA
<213> 人工序列(Artificial Sequence)
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gatgtaatcc ttgccgtcgt cggtta 26
<210> 17
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
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gcgatacttc tatcaccaag aggactc 27
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<212> DNA
<213> 人工序列(Artificial Sequence)
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gctctccaca accccgta 18
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<213> 人工序列(Artificial Sequence)
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acttgtgctt ggtggtctga gct 23
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<213> 人工序列(Artificial Sequence)
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aaagtgaagg cccactcaca gact 24
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<213> 人工序列(Artificial Sequence)
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gacccgccca aaccctcgac c 21
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tgggaccgga acacacagat 20
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cgacctgggg cccgacg 17
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<213> 人工序列(Artificial Sequence)
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cgggccgagg tggacac 17
Claims (7)
1.HLA亚型检测试剂盒,其特征在于,所述试剂盒包括用于检测多种HLA亚型的多重PCR扩增的引物和探针,每对HLA亚型上游和下游引物的量比值为1:20到1:50,每对引物上游引物的Tm值比下游引物的Tm值高4-8℃。
2.根据权利要求1所述的试剂盒,其特征在于,每对HLA亚型的上游和下游引物的量比值为1:30到1:40。
3.根据权利要求1所述的试剂盒,其特征在于,在所述上游引物5’端增加碱基GCG以提高所述上游引物的Tm值。
4.根据权利要求1所述的试剂盒,其特征在于,所述试剂盒是用于检测以下HLA亚型的试剂盒:HLA-A*02:01,HLA-A*24:02,HLA-B*15:01,HLA-B*15:02,HLA-B*15:11,HLA-B*35:01,HLA-B*38:02,HLA-B*13:01,HLA-DRB1*04:06。
7.根据权利要求1-6任一所述的试剂盒在癫痫用药过敏预警中的应用。
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