CN112538527B - 用于检测人类白细胞抗原b位点1502基因的引物和探针组合及试剂盒 - Google Patents
用于检测人类白细胞抗原b位点1502基因的引物和探针组合及试剂盒 Download PDFInfo
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Abstract
本发明涉及体外诊断技术领域,特别涉及用于检测人类白细胞抗原B位点1502基因的引物和探针组合及试剂盒。该引物和探针组合包括扩增第5内含子的引物和探针以及扩增第2外显子的引物和探针;扩增第5内含子的引物由SEQ ID NO:1所示正向引物和SEQ ID NO:2所示反向引物组成;扩增第5内含子的探针序列如SEQ ID NO:3所示;扩增第2外显子的引物由SEQ ID NO:4所示正向引物和SEQ ID NO:5所示反向引物组成;扩增第2外显子的探针序列如SEQ ID NO:6所示。本发明提供的实时荧光定量PCR的引物和探针灵敏度高、特异性强,可准确检出低至1ng/ul的基因组DNA。
Description
技术领域
本发明涉及体外诊断技术领域,特别涉及用于检测人类白细胞抗原B位点1502基因的引物和探针组合及试剂盒。
背景技术
癫痫又称脑痫、羊角风,是一种由多种病因引起的慢性脑部疾病,以脑神经元过度放电导致反复性、发作性和短暂性的中枢神经系统功能失常为特征。抗癫痫药物治疗是癫痫治疗最重要和最基本的治疗,也往往是癫痫的首选治疗。癫痫在任何年龄、地区和种族的人群中都有发病,但以儿童和青少年发病率较高。近年来随着我国人口老龄化,脑血管病、痴呆和神经系统退行性疾病的发病率增加,老年人群中癫痫发病率已出现上升的趋势。国内流行病学资料显示,我国癫痫的患病率在4‰到7‰之间,每年近40万新发癫痫患者。癫痫患者的死亡危险性为一般人群的2-3倍,已成为神经科仅次于脑血管病的第二大常见疾病。WHO将癫痫列为重点防控的神经、精神疾病。
卡马西平(Carbamazeine)是临床精神科常用的一线抗癫痫药物。但其治疗窗窄,血药浓度个体差异大,易产生药物不良反应(Adverse Drug Reactions,ADRs),包括严重的皮肤不良反应(Severe Cutaneous Adverse reactions,SCARs),如史蒂文斯-约翰逊综合征(Stevens-Johnson Syndrome,SJS)/中毒性表皮坏死溶解症(Toxic EpidemnalNecrolysis,TEN)等。药物不良反应就是合格的药品在正常的用法用量下出现的与用药目的无关的意外的有害反应。其中罕见、严重的不良反应一般与用药人特异体质因素或某些代谢酶的遗传因素有关。
SJS又称重症多形红斑,特点是大面积表皮和黏膜上皮细胞的脱落,且口咽部、眼睛、生殖器和肛门等黏膜组织也都有典型表现;TEN则为更严重的类型,有研究认为它们是同一种疾病的两种不同严重程度。当皮肤剥离面积小于10%为SJS,大于30%为TEN,两者之间则为SJS/TEN重叠。SJS与TEN每年的发病率分别为1-6/106和1-2/106,但致死率很高,尤其TEN较为严重,致死率高达40%。虽然由芳香族抗癫痫药物诱发的严重皮肤不良反应发病率较低,但是因其严重威胁患者的生命,越来越引起足够的重视。有研究证实,卡马西平诱导的SJS/TEN与患者是否携带HLA-B*1502等位基因有很大关联。
HLA-B*1502是一种人类白细胞抗原HLA等位基因,几乎只存在于亚洲人种之中,包括中国汉族人、菲律宾人、马来西亚人、南亚印度人和泰国人。人类白细胞抗原(humanleucocyte antigen,HLA)是人类主要组织兼容性抗原(major histocomptibilityantigen,MHC)的别称,HLA基因位于6号染色体短臂上的21.31区(6p21.31),是一群紧密连锁的复等位基因,包括200多个基因座位,共554个等位基因,全长4079kb,占人类整个基因组的1/3000,是最为复杂的调控人体特异性免疫应答和决定疾病易感性个体差异的主要基因系统,在不同的种族或同一种族不同群体中的分布显示明显的特异性。根据HLA基因表达产物的结构、表达方式、组织分布与功能不同,至少可将其分为3类,即HLA-I类、II类及III类基因。
目前检测HLA-B*1502的主要方法为PCR-SBT(sequence based typing,基于测序分型的聚合酶链反应)、PCR-SSP(sequence specific primer,序列特异引物引导的聚合酶链反应)、PCR-SSO(sequence specifie oligonucleotide,序列特异性寡核苷酸探针杂交聚合酶链反应)和RT-PCR(荧光定量PCR反应)等检测方法。
在现有检测方法中,PCR-SBT方法是现世界卫生组织(WHO)推荐的HLA分型方法的“金标准”,是对HLA基因的B位点进行测序,进而确定HLA-B*1502高分辨率的方法,但其检测的步骤繁琐,需要提供精密仪器,费用昂贵并且周转时间较长(大于1天),不适合对于用药安全性的快速指导。PCR-SSP方法是利用与HLA等位基因序列互补的引物,对待测样本DNA进行特异性PCR扩增,并用琼脂糖凝胶电泳检测PCR产物,操作过程复杂,耗时也较长。PCR-SSO即多聚酶链反应寡聚核苷酸探针杂交,先采用位点特异性引物对HLA-B位点进行扩增,扩增产物包括HLA-B位点的所有等位基因序列,再根据碱基互补原则,将PCR产物经化学变性解链后,单链与固化在2条尼龙膜上的序列特异寡核苷酸探针在特定条件下杂交,经洗膜,加入标记有碱性磷酸盐的抗生物蛋白链菌素与生物素及底物反应,对扩增片段进行分析鉴定,但其耗时长,需严格控制实验条件,否则会导致错配,影响结果准确性。虽然,总的来说PCR-SSP是一种相对简单方便、特异性高的方法,且目前已有基于PCR-SSP方法进行单特异引物检测HLA-B*1502的试剂盒,但是目前市面上的试剂盒涵盖的基因型别较少,容易出现假阳性,远远不能满足对用药安全性的快速、简便、高特异性检测HLA-B*1502基因的需求。因此,本领域急需一种操作简便、快速,且准确度高的检测方法。
发明内容
有鉴于此,本发明提供了用于检测人类白细胞抗原B位点1502基因的引物和探针组合及试剂盒。本发明提供的实时荧光定量PCR的引物和探针灵敏度高、特异性强,可准确检出低至1ng/μL的基因组DNA。本发明还提供了一种检测试剂盒,采用单管扩增,操作简便,特异性强,可配合自动化仪器检测。
为了实现上述发明目的,本发明提供以下技术方案:
本发明提供了用于检测人类白细胞抗原B位点1502基因的引物和探针组合,包括扩增第5内含子的引物和探针以及扩增第2外显子的引物和探针;
扩增第5内含子的引物由SEQ ID NO:1所示正向引物和SEQ ID NO:2所示反向引物组成;扩增第5内含子的探针序列如SEQ ID NO:3所示;
扩增第2外显子的引物由SEQ ID NO:4所示正向引物和SEQ ID NO:5所示反向引物组成;扩增第2外显子的探针序列如SEQ ID NO:6所示。
作为优选,引物和探针组合还包括内标引物和内标探针,内标引物由SEQ ID NO:7所示正向引物和SEQ ID NO:8所示反向引物组成;内标探针序列如SEQ ID NO:9所示。
作为优选,引物和探针组合中的探针为Taqman探针,探针的核苷酸序列5’端标记荧光基团,3’端标记荧光淬灭基团。
作为优选,荧光基团为FAM、ROX、CY5中的一种,扩增第5内含子的探针、扩增第2外显子的探针与内标探针5’端标记不同的荧光基团。
作为优选,引物和探针组合中的探针为MGB探针。
本发明还提供了用于检测人类白细胞抗原B位点1502基因的试剂盒,包括PCR反应液1和PCR反应液2;PCR反应液1包括上述引物和探针组合,以及PCR缓冲液、dNTPs、甘油、rTth酶、UDG酶、防腐剂和水;
PCR反应液2为醋酸锰水溶液。
作为优选,PCR反应液1中各组分浓度为:
PCR缓冲液为含有Tricine和醋酸钾的水溶液;Tricine的浓度为0.04~0.06M,pH值为8.0~9.0;醋酸钾的浓度为0.09~0.12M。
作为优选,PCR反应液2中醋酸锰的浓度为7.5~22.5mmol/L。
作为优选,检测人类白细胞抗原B位点1502基因的PCR反应体系为:
PCR反应液1 5~50μL
PCR反应液2 5~50μL
样本 10~100μL;
扩增程序为:50℃2min;95℃2min;95℃10s,60℃22s,45个循环;40℃10s。
作为优选,试剂盒还包括阴性质控和阳性质控。
在本发明提供的具体实施例中,防腐剂为叠氮化钠。
本发明提供了用于检测人类白细胞抗原B位点1502基因的引物和探针组合及试剂盒。该引物和探针组合包括扩增第5内含子的引物和探针以及扩增第2外显子的引物和探针;扩增第5内含子的引物由SEQ ID NO:1所示正向引物和SEQ ID NO:2所示反向引物组成;扩增第5内含子的探针序列如SEQ ID NO:3所示;扩增第2外显子的引物由SEQ ID NO:4所示正向引物和SEQ ID NO:5所示反向引物组成;扩增第2外显子的探针序列如SEQ ID NO:6所示。
本发明的试剂盒具有以下优点:
1.本发明试剂盒通过设计2对特异性引物扩增HLA-B基因的第二号外显子和第五号内含子,从而对癫痫患者是否具有HLA-B*1502基因进行的检测,即采用多重PCR扩增,一次检测可以准确检测单个样本的两个特异性区的基因型,准确度高;
2.操作简单,单管PCR即可对单个样本的的两个特异性区进行准确分型;
3.检测灵敏度高,可以准确检测低至1ng/μL基因组浓度的DNA的基因型;
4.针对HLA-B*1502基因的两个特异性区分别设计对应的ARMS引物和Taqman探针,可以特异性扩增识别对应的多态性;
5.使用荧光定量PCR技术进行检测,检测过程引入UDG酶防污染体系,且全程闭管,因此可以大大降低污染风险,保证检测的准确性;
6.快速检测,整个检测过程在50分钟内完成,判读方法简单可行;
7.PCR试剂实现4℃保存,避免传统-20℃保存,经反复冻融后试剂性能下降的风险,同时提高了用户的使用便捷性。
8.本试剂盒包含了HLA-B*1502的两个特异性区的基因型检测,可用于特异性检测HLA-B*1502等位基因。本发明对HLA-B*1502基因的定性检测,可配合自动化仪器检测,为临床指导卡马西平用药提供参考,适于推广应用。
附图说明
图1-图5分别是HLA-B*1502基因的两个特异区两种基因型的扩增检测图;
图1HLA-B*1502阳性质控;
图2HLA-B*1502阴性质控;
图3HLA-B*1502阳性样本;
图4HLA-B*1502阴性样本1;
图5HLA-B*1502阴性样本2。
具体实施方式
本发明公开了用于检测人类白细胞抗原B位点1502基因的引物和探针组合及试剂盒,本领域技术人员可以借鉴本文内容,适当改进工艺参数实现。特别需要指出的是,所有类似的替换和改动对本领域技术人员来说是显而易见的,它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述,相关人员明显能在不脱离本发明内容、精神和范围内对本文所述的方法和应用进行改动或适当变更与组合,来实现和应用本发明技术。
Taqman荧光定量PCR法操作简便、快速,特异性高,并可实现高通量检测。一般来讲,Taqman荧光定量PCR法通过设计一对或多对HLA-B*1502特异性引物及相应探针,进行PCR扩增,考虑到HLA多态性非常高,结果易出现假阳性,从而影响其准确性。因此,本发明克服现有技术中的不足,在Taqman探针的基础上,提供一种操作简便、高灵敏度、特异性强的人类白细胞抗原B位点1502基因检测引物和探针,本发明的另一目的是提供包括检测以上等位基因的试剂盒。
所述探针均为Taqman探针,探针的核苷酸序列5’端标记发光基团,3’端标记荧光淬灭基团,且HLA-B*1502两个特异区靶标和内标三个探针序列分别标记不同的Taqman探针荧光基团。
优选的,HLA-B*1502第一特异区的发光基团标记为FAM,HLA-B*1502第二特异区的发光基团标记为ROX,内标的发光基团标记为CY5。
优选的,所述人类白细胞抗原B位点1502基因检测试剂盒,还包括PCR反应液,PCR反应液包括dNTPs、PCR缓冲液、甘油、超纯水、rTth酶、UDG防污染酶、金属离子K+、叠氮化钠等。
优选的,所述人类白细胞抗原B位点1502基因检测试剂盒,还包括阴性质控和阳性质控。
本发明还提供了上述人类白细胞抗原B位点1502基因检测试剂盒的检测方法,包括以下步骤:
1、样本制备:分离纯化样品中的基因组DNA;
2、反应液配制:用本发明的人类白细胞抗原B位点1502基因检测引物探针及PCR试剂配制的PCR反应液1、PCR反应液2构建PCR反应体系,每个反应体系均用DNA作为模板,进行荧光定量PCR,同时设置阴性质控和阳性质控;
1)收集荧光信号:选择对应荧光基团的荧光检测模式,基线调整取3~15个循环的荧光信号,阈值线设定以阈值线刚好超过正常阴性质控品扩增曲线(无规则的噪音线)的最高点为原则。
2)检测结果分析:阳性质控荧光扩增曲线均超过阈值线,有明显的扩增且FAM、ROX、Cy5信号Ct≤35,阴性质控Cy5信号有明显的扩增且Cy5信号Ct≤35,FAM、ROX信号无明显的扩增且Ct≥35或无信号值;待检测样本Cy5信号有明显的扩增且产生的Cy5信号的荧光扩增曲线均超过阈值线,表明扩增正常,按照下面标准进行基因型判断:
3、待测样本检测结果判读:
FAM和ROX均有明显的扩增曲线,FAM信号的Ct值≤35,ROX信号的Ct值≤35为阳性;
FAM通道没有明显的扩增曲线,或FAM通道信号的Ct值>35为阴性。
本发明的人类白细胞抗原B位点1502基因检测试剂盒采用Taqman探针法,建立了在单管反应检测双靶标的多重荧光定量PCR方法。本发明的HLA-B*1502基因检测试剂盒采用ARMS引物特异性扩增目的片段,同时采用普通Taqman探针以降低成本,同时引入内标体系和UDG酶防污染系统,更加准确、稳定的对样本进行分型检测。
本发明提供的用于检测人类白细胞抗原B位点1502基因的引物和探针组合及试剂盒中所用试剂或仪器均可由市场购得。
下面结合实施例,进一步阐述本发明:
实施例1:试剂盒的组成
本发明所述试剂盒的组成如下表:
表1
其中,PCR反应液1各组分浓度为:
PCR反应液1中的PCR缓冲液为:0.05M的Tricine(8.3),0.1M的醋酸钾,水;
PCR反应液2中的醋酸锰的浓度为20.5mmol/L。
特异性引物和探针、内标引物和探针序列如下:
1、第5内含子引物和探针:
F1:5’-GACATTCACTGGGAAGCAGCAGTCT-3’
R1:5’-GTTGGACTGGGAGGTAGCATGA-3’
P1:5’-FAM-ACGTAGCCTGGGACCCTCTGTG-BHQ1-3’
2、第2外显子引物和探针:
F2:5’-TTCGACAGCGACGCCG-3’
R2:5’-GGTAGTAGTAGCTGCGCAG-3’
P2:5’-ROX-TTCCGCAGGCTCGCTCGGTAGGTCTG-BHQ2-3’
3、内标引物和探针分别为:
F3:5’-GAAGGCTCATGGCAAGAAAG-3’
R3:5’-CTCACTCAGTGTGGCAAAGG-3’
P3:5’-Cy5-CTTGAGGTTGTCCAGGTGAGCCAG-BHQ2-3’
采用两对引物探针对双靶标扩增,减少假阳性,增强特异性。
实施例2:样本基因组DNA的提取
本实施例中收集人全血/外周血样本,从中提取基因组DNA。
从EDTA抗凝血浆中提取基因组DNA,用其作为PCR检测的模板。采用郑州安图生物工程股份有限公司的前处理试剂和核酸提取纯化试剂(磁珠法),按照说明进行,具体详述如下:
1.样本前处理:
(1)冻融全血用红细胞裂解液进行前处理:
①取200μL临床冻融或发生溶血全血样本,加入600μL红细胞裂解液,轻轻颠倒混匀10次,红细胞裂解后,溶液应该是清亮透明的。12000rpm,离心3min,弃上清;
②加入600μL 0.2mg/mL蛋白酶K,常温20-25℃孵育5min后,12000rpm离心3min,弃上清,取沉淀;
③加入600μL裂解液涡旋充分重悬沉淀,备用。
(2)非冻融全血用淋巴细胞分离液处理:
①取200μL EDTA抗凝血缓慢加入含有600μL淋巴细胞分离液EP管中(不可晃动,直接离心)3000rpm,离心5min,此时离心管中由上至下细胞分四层(第一层:为血浆层。第二层:为环状乳白色淋巴细胞层。第三层:为透明分离液层。第四层:为红细胞层)。
②取前三层上清液备用。
2.样本提取:
①将10μL蛋白酶K、20μL磁珠混悬液(吸前振荡混匀磁珠)、1.3mL裂解液(pH2.0-5.0,浓度40%-60%的胍盐)、600μL血清/血浆样本或处理后其他样本类型样本液依次加入5mL离心管,混匀,于37℃培养箱中孵育2min;
②将孵育后的上述混合液转移至2mL离心管中,放置在磁力架上,磁吸2min,弃上清;
③去除磁力架,添加2mL洗液A(胍盐浓度1-3M,Tris-HCl浓度为10-80mM),混匀,放置在磁力架上,磁吸2min,弃上清;
④去除磁力架,添加2mL洗液B(Tris-HCl浓度为5-60mM,Nonidet P40浓度为0.04%-0.8%),混匀,放置在磁力架上,磁吸2min,弃上清(尽量减少残留);
⑤加100-300μL(按照后续实验要求添加)洗脱液(Tris-base(pH7.0-9.0)),振荡混匀,于80℃干式恒温器中解离5min;
⑥放置在磁力架上,磁吸2min后,取上清液用于后续实验。
实施例3:样本基因组DNA的检测
本实施例采用实施例1中所列举的引物、探针组合来扩增实施例2中所提取的基因组DNA样品。检测步骤如下:
①构建反应体系
计算所需要的样本数,按照下表中的量等比例增加试剂量,阴阳性质控只做一份。
表2
②上机检测
直接上荧光定量PCR仪进行检测,扩增程序设置为:
表3
每个循环的最后收集FAM、ROX、CY5荧光信号。
③检测结果分析
表4
实施例4
依据现有专利CN104450914A、CN104830852B、CN105296615A、CN106119362B、CN106520971A中列出的引物探针组合序列与本专利引物探针组合序列进行对比分析如下表5-7所示:
表5现有专利与本专利正向引物序列
注:代表序列相同。
表6现有专利与本专利反向引物序列
表7现有专利与本专利荧光探针序列
综上,本发明除与专利CN106520971A正向引物有部分序列重叠一致外,其余与对比专利均无序列重叠一致,且本专利采用Taqman探针。
实施例5对比试验
依据现有专利CN104450914A、CN104830852B、CN105296615A、CN106119362B、CN106520971A中的方案,配制成PCR反应液方案1、方案2、方案3、方案4、方案5,与本研究实施例1进行对比:
实验一:灵敏度对比
按照实施例2提取临床样本基因组DNA;将已提取的DNA使用郑州安图生物工程股份有限公司的核酸提取或纯化试剂配套洗脱液稀释至0.5ng/μL、0.1ng/μL、0.05ng/μL、0.025ng/μL、0.01ng/μL浓度进行检测,各方案对每个浓度均重复检测21次,具体数据见表8。
表8:HLA-B*1502阳性检出率
综上,检测HLA-B*1502的最低检出限浓度样本(0.1ng/μL)时,本研究方案均能够全部检出,检出率100%,且在Ct值的重复性上均优于现有专利方案1、方案2、方案3、方案4、方案5。
实验二:特异性对比
将5个方案的PCR反应试剂分别检测收集的HLA-B*1502阴性临床样本100例,阳性临床样本84例与测序进行临床符合率比对,结果如表9:
表9:HLA-B*1502临床检测结果
结果 | 实施例1 | 方案1 | 方案2 | 方案3 | 方案4 | 方案5 |
阳性 | 84 | 86 | 85 | 88 | 92 | 87 |
阴性 | 100 | 98 | 99 | 96 | 92 | 97 |
阳性率 | 100% | 97.67% | 98.82% | 95.45% | 91.30% | 96.55% |
阴性率 | 100% | 100.00% | 100.00% | 100.00% | 100.00% | 100.00% |
与测序一致率 | 100% | 98.91% | 99.46% | 97.83% | 95.65% | 98.37% |
综上结果表明,检测HLA-B*1502临床样本进行分型验证时,本发明与测序法的符合率达到100%,符合率明显优于现有专利方案1、方案2、方案3、方案4、方案5。说明本发明设计的引物探针特异性强,无假阳性。
以上所述仅是本发明的优选实施方式,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的保护范围。
序列表
<110> 郑州安图生物工程股份有限公司
<120> 用于检测人类白细胞抗原B位点1502基因的引物和探针组合及试剂盒
<130> MP2013957
<160> 9
<170> SIPOSequenceListing 1.0
<210> 1
<211> 25
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 1
gacattcact gggaagcagc agtct 25
<210> 2
<211> 22
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 2
gttggactgg gaggtagcat ga 22
<210> 3
<211> 22
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 3
acgtagcctg ggaccctctg tg 22
<210> 4
<211> 16
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 4
ttcgacagcg acgccg 16
<210> 5
<211> 19
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 5
ggtagtagta gctgcgcag 19
<210> 6
<211> 26
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 6
ttccgcaggc tcgctcggta ggtctg 26
<210> 7
<211> 20
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 7
gaaggctcat ggcaagaaag 20
<210> 8
<211> 20
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 8
ctcactcagt gtggcaaagg 20
<210> 9
<211> 24
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 9
cttgaggttg tccaggtgag ccag 24
Claims (9)
1.用于检测人类白细胞抗原B位点1502基因的引物和探针组合,其特征在于,包括扩增第5内含子的引物和探针以及扩增第2外显子的引物和探针;
所述扩增第5内含子的引物由SEQ ID NO:1所示正向引物和SEQ ID NO:2所示反向引物组成;所述扩增第5内含子的探针序列如SEQ ID NO:3所示;
所述扩增第2外显子的引物由SEQ ID NO:4所示正向引物和SEQ ID NO:5所示反向引物组成;所述扩增第2外显子的探针序列如SEQ ID NO:6所示;
所述引物和探针组合中的探针为Taqman探针,探针的核苷酸序列5’端标记荧光基团,3’端标记荧光淬灭基团。
2.根据权利要求1所述的引物和探针组合,其特征在于,所述引物和探针组合还包括内标引物和内标探针,所述内标引物由SEQ ID NO:7所示正向引物和SEQ ID NO:8所示反向引物组成;所述内标探针序列如SEQ ID NO:9所示。
3.根据权利要求2所述的引物和探针组合,其特征在于,所述荧光基团为FAM、ROX、CY5中的一种,扩增第5内含子的探针、扩增第2外显子的探针与内标探针5’端标记不同的荧光基团。
4.根据权利要求1或2所述的引物和探针组合,其特征在于,所述引物和探针组合中的探针为MGB探针。
5.用于检测人类白细胞抗原B位点1502基因的试剂盒,其特征在于,包括PCR反应液1和PCR反应液2;所述PCR反应液1包括权利要求1至4中任一项所述的引物和探针组合,以及PCR缓冲液、dNTPs、甘油、rTth酶、UDG酶、防腐剂和水;
所述PCR反应液2为醋酸锰水溶液。
6.根据权利要求5所述的试剂盒,其特征在于,所述PCR反应液1中各组分浓度为:
引物 0.1~0.3μM
探针 0.1~0.2μM
PCR缓冲液 30vt%~70vt%
dNTPs 100~300μM
甘油 2.0vt%~25vt%
rTth酶 1~5U/μL
UDG酶 0.01~0.05U/μL
防腐剂 0.05w/v%~0.15w/v%
水 补足;
所述PCR缓冲液为含有Tricine和醋酸钾的水溶液;所述Tricine的浓度为0.04~0.06M,pH值为8.0~9.0;所述醋酸钾的浓度为0.09~0.12M。
7.根据权利要求5或6所述的试剂盒,其特征在于,所述PCR反应液2中醋酸锰的浓度为7.5~22.5mmol/L。
8.根据权利要求7所述的试剂盒,其特征在于,所述检测人类白细胞抗原B位点1502基因的PCR反应体系为:
PCR反应液1 5~50μL
PCR反应液2 5~50μL
样本 10~100μL;
扩增程序为:50℃ 2min;95℃ 2min;95℃ 10s,60℃ 22s,45个循环;40℃ 10s。
9.根据权利要求5至8中任一项所述的试剂盒,其特征在于,所述试剂盒还包括阴性质控和阳性质控。
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