CN112608990A - 检测ABCB4 c.3508-16T>C位点突变的引物和方法 - Google Patents
检测ABCB4 c.3508-16T>C位点突变的引物和方法 Download PDFInfo
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Abstract
本发明涉及检测ABCB4 c.3508‑16T>C碱基突变的引物,包括扩增ABCB4 c.3508‑16T>C位点的引物;采用Sanger测序技术,可用于快速检测进行性家族性胆汁淤积(PFIC,progressive familial intrahepatic cholestasis)III型患者体内ABCB4 c.3508‑16T>C位点突变情况。利用本发明完成的检测结果准确,对PFIC III的临床鉴别诊断有重要的参考意义。
Description
技术领域
本发明属于生命科学和生物技术领域,特别涉及检测ABCB4 c.3508-16T>C,位点突变的引物,采用普通PCR结合Sanger测序技术,可用于快速检测进行性家族性胆汁淤积III型患者体内ABCB4 c.3508-16T>C位点的突变情况。
背景技术
家族性胆汁淤积是以家族遗传为特点的慢性复发性胆汁淤积综合征,是遗传性的消化系统疾病。此病发病率约1/100000,呈世界性分布,男女发病率无明显差异。家族性胆汁淤积分为进行性家族性肝内胆汁淤积症(progressive familial intrahepaticcholestasis,PFIC)和良性复发性肝内胆汁淤积症(benign recurrent intrahepaticcholestasis,BRIC)。PFIC是一种由基因突变所致胆汁分泌或排泄障碍而形成的胆汁淤积症,为常染色体隐性遗传疾病,根据突变基因不同分为3型,分别为PFIC I型(PFIC I)、PFICII型(PFIC II)和PFIC III型(PFIC III)。PFIC III由ABCB4(ATP binding cassettesubfamily B member 4)基因突变引起。临床上可表现为突出的和特征性的瘙痒、反复发作性的高结合胆红素血症、白陶土样便、肝脾大,因肝硬化门脉高压可发生胃肠出血,常在成年前进展为肝硬化和肝衰竭。PFIC III与PFIC I、PFIC II的主要区别在于血清γ-谷氨酰转肽酶(GGT)升高及肝组织病理表现为明显的小胆管增生。
ABCB4基因,又称为PGY3或MDR3,位于人类第7条常染色体长臂2区1带1亚(7q21.1),跨距74kb,cDNA全长4.1kb,包括28个外显子,其中27个外显子具有编码序列。ABCB4主要在肝细胞胆管膜上有高度表达,此外在心肌细胞、骨骼肌细胞和脾脏B淋巴细胞中有较低程度的表达。ABCB4基因编码MDR3蛋白,该蛋白是一种磷脂转运蛋白,位于肝细胞毛细胆管膜上,为磷脂输出泵,将磷脂从肝细胞转运到胆管中,是胆管中磷脂分泌的限速步骤。MDR3蛋白共分4个区:2个核苷酸结合区(nucleotide binding domain,NBD)和2个同源的跨膜区(transmembrane domain,TMD)。正常情况下,肝细胞合成的磷脂通过MDR3转运到胆汁中,与胆盐共同形成微粒,使胆盐亲水性增加,减轻胆盐的去垢作用,保护胆管细胞免受胆盐的毒性损伤。当ABCB4发生突变后,导致与胆管中磷脂分泌相关的MDR3蛋白缺失或表达降低,胆汁中磷脂缺乏,胆盐不能与磷脂构建混合微粒,胆盐游离,对毛细胆管膜发生毒性去垢作用,使胆管细胞发生损伤,出现胆汁淤积、小胆管增生、炎症浸润,逐渐进展为门管区纤维化,肝硬化及门静脉高压,最后发展为终末期肝病。
目前与ABCB4基因突变有关的疾病已有很多,包括PFIC III,胆石症,药物性肝内胆汁淤积症和原发性胆汁性肝硬化等,同时已报道的ABCB4基因突变类型也有很多,包括无义突变、错义突变、缺失突变和小片段碱基的插入。其中,国外研究报道显示,PFIC III在一些有近亲婚配宗教习俗的人群中,发病率稍高。另外,在北非、意大利、土耳其等欧洲白人、中国大陆与中国台湾也有相关报道,且均为散发报道,尚未发现热点突变。中国人口基数大,可能存在众多PFIC患者,因此本发明针对PFIC III采用PCR产物直接测序法对ABCB4基因的全外显子进行检测,通过对其进行突变分析,初步探讨其基因突变特点,有助于更加全面了解基因突变的发生情况及其位点,有助于提高诊断水平和认识,为遗传咨询奠定基础,为PFIC III的早期诊断及治疗提供新的方法。
发明内容
本发明的目的是提供一种检测ABCB4 c.3508-16T>C位点突变的引物,采用PCR技术,可用于快速检测PFIC III患者体内ABCB4 c.3508-16T>C位点的突变情况,包括扩增ABCB4 c.3508-16T>C位点的引物,其碱基序列为:ABCB4-A-F:
TGTAAAACGACGGCCAGTTGTAAATAGAACTGTCAACTGTTAAGC
ABCB4-A-R:AACAGCTATGACCATGTTTTCATGGTTGACAGCAAAATC。
优选地,还包括测序引物,其碱基序列为:
M13F:TGTAAAACGACGGCCAGT
M13R:AACAGCTATGACCATG。
优选地,ABCB4-A-F和ABCB4-A-R的摩尔浓度比1:1。
优选地,M13F和M13R的摩尔浓度比1:1。
本发明还提供了一种检测ABCB4 c.3508-16T>C位点突变情况的方法,包括以下步骤:
(1)提取血液基因组DNA;
(2)利用一对PCR扩增引物扩增步骤(1)中提取的DNA,获取扩增产物;
(3)利用一对测序引物对步骤(2)中的扩增产物进行测序;
(4)对测序结果进行判断,确定ABCB4 c.3508-16T>C位点是否发生突变;其中所述一对PCR扩增引物的碱基序列为:
ABCB4-A-F:
TGTAAAACGACGGCCAGTTGTAAATAGAACTGTCAACTGTTAAGC
ABCB4-A-R:AACAGCTATGACCATGTTTTCATGGTTGACAGCAAAATC;
所述一对测序引物的碱基序列为:
M13F:TGTAAAACGACGGCCAGT
M13R:AACAGCTATGACCATG。
本发明还提供了一种检测ABCB4 c.3508-16T>C位点突变的试剂盒,所述试剂盒包括:(i)血液基因组DNA抽提试剂;
(ii)检测体系PCR扩增反应液:包括扩增ABCB4 c.3508-16T>C位点的引物,其碱基序列为:
ABCB4-A-F:
TGTAAAACGACGGCCAGTTGTAAATAGAACTGTCAACTGTTAAGC
ABCB4-A-R:AACAGCTATGACCATGTTTTCATGGTTGACAGCAAAATC
(iii)阳性对照品和阴性对照品。
优选地,所述试剂盒还包括测序体系试剂:包括测序引物,其碱基序列为:M13F:TGTAAAACGACGGCCAGT
M13R:AACAGCTATGACCATG。
有益效果:本发明设计了扩增ABCB4 c.3508-16T>C的引物。采用PCR技术,构建了稳定的扩增体系。通过调整引物浓度、退火温度等反应条件,可使扩增效率达到最佳。本发明利用测序技术检测患者ABCB4 c.3508-16T>C突变热点的方法,可以将ABCB4基因ABCB4c.3508-16T>C的突变检测出来,相比较荧光定量PCR法降低了检测的成本和难度。荧光定量PCR法要针对不同的突变类型设计3个探针,成本高,检测难度大。
附图说明
图1为20例样本验证的琼脂糖凝胶电泳结果(1-20表示样本序号,M为Marker DL2000),PCR扩增后,引物扩增有效,而且目的条带清晰,产物大小正确。
图2 13号样本ABCB4 c.3508-16位点碱基T突变为碱基C测序截图演示。
具体实施方式
实施例1
下面结合具体实施例和附图,进一步阐述本发明。应当注意的是,实施例中未说明的常规条件和方法,通常按照所属领域实验人员常规采用方法:譬如,奥斯柏和金斯顿主编的《精编分子生物学实验指南》第四版,或者按照制造厂商所建议的步骤和条件。
一种检测ABCB4 c.3508-16T>C位点突变的引物,包括:
扩增ABCB4 c.3508-16T>C位点碱基的引物,其碱基序列为:
ABCB4-A-F:
TGTAAAACGACGGCCAGTTGTAAATAGAACTGTCAACTGTTAAGC
ABCB4-A-R:AACAGCTATGACCATGTTTTCATGGTTGACAGCAAAATC
一种检测ABCB4 c.3508-16T>C位点突变的试剂盒,包括:
(i)血液DNA抽提试剂;
(ii)检测体系PCR反应液;
(iii)测序体系试剂;
(iv)阳性对照品和阴性对照品。
其中,血液基因组DNA抽提试剂购自天根血液基因组DNA抽提试剂盒等商品化试剂。
检测体系PCR扩增反应液包括:2×PCR Buffer;2mM dNTPs;KOD FX DNAPolymerase(1U/μL);扩增ABCB4 c.3508-16T>C位点的上游引物ABCB4-A-F(10μM)、下游引物ABCB4-A-R(10μM)。
测序体系试剂包括:测序纯化液(ExoI:0.6U,CIP:1.2U)、EDTA(125mmo l)、无水乙醇、75%乙醇、HIDI(高度去离子甲酰胺)、测序引物:对上游引物A BCB4-A-F和下游引物ABCB4-A-R的扩增产物进行测序的引物M13F(3.2μM)和M13R(3.2μM)。
实施例2
(1)血液基因组DNA抽提试剂盒(天根生物DP318-03)的操作流程:
1)抽取500μl血液加入1000μl红细胞裂解液,颠倒混匀,室温放置5分钟,期间再颠倒混匀几次。12,000rpm离心1min,吸去上清,留下白细胞沉淀,加200μl缓冲液GA,振荡至彻底混匀。
2)加入20μl蛋白酶K溶液,混匀。
3)加入200μl缓冲液GB,充分颠倒混匀,70℃放置10分钟,溶液应变清亮,简短离心以去除管盖内壁的水珠。
4)加入200μl无水乙醇,充分振荡混匀15秒,此时可能会出现絮状沉淀,简短离心以去除管盖内壁的水珠。
5)将上一步所得溶液和絮状沉淀都加入一个吸附柱CB3中(吸附柱放入收集管中),12,000rpm离心30秒,倒掉废液,将吸附柱CB3放回收集管中。
6)向吸附柱CB3中加入500μl缓冲液GD(使用前请先检查是否已加入无水乙醇),12,000rpm离心30秒,倒掉废液,将吸附柱CB3放入收集管中。
7)向吸附柱CB3中加入700μl漂洗液PW(使用前请先检查是否已加入无水乙醇),12,000rpm离心30秒,倒掉废液,将吸附柱CB3放入收集管中。
8)向吸附柱CB3中加入500μl漂洗液PW,12,000rpm离心30秒,倒掉废液。
9)将吸附柱CB3放回收集管中,12,000rpm离心2分钟,倒掉废液。将吸附柱CB3置于室温放置数分钟,以彻底晾干吸附材料中残余的漂洗液。
10)将吸附柱CB3转入一个干净的离心管中,向吸附膜的中间部位悬空滴加100μl洗脱缓冲液TE,室温放置2-5分钟,12,000rpm离心2分钟,将溶液收集到离心管中。
(2)试剂配置:按检测人份数配置检测体系PCR反应液各Xμl,每人份18μl分装:
X=18μl反应液×(n份标本+1份阳性对照+1份阴性对照+1份空白对照)
n为检测标本数。
(3)加样:加入检测体系PCR反应液中2μl DNA;阳性对照和阴性对照直接加2μl阳性对照品和阴性对照品;空白对照加2μl生理盐水或不加任何物质。
(4)扩增:检测在常规PCR仪上进行,可用仪器包括ABI veriti(美国AppliedBiosystems公司)等。反应条件如下:
PCR扩增体系试剂配制方法如下:
其中,引物序列为:
ABCB4-A-F:TGTAAAACGACGGCCAGTTGTAAATAGAACTGTCAACTGTTAAGC
ABCB4-A-R:AACAGCTATGACCATGTTTTCATGGTTGACAGCAAAATC
(5)电泳:1.5%琼脂糖凝胶电泳,110V,35min,凝胶成像系统观察。
如图1所示,即为1例血液样本以相应的引物扩增后所得产物的电泳图谱。通过电泳图的分析表明本发明所述扩增有效,且条带清晰。
(6)Sanger测序:
取9 PCR产物与2μl纯化体系。按照以下程序进行纯化:
将1μl纯化产物分别与上、下测序引物按照如下体系进行混合:
测序反应程序:
沉淀环节:
向完成测序反应的产物中加入2μl 125mmol的EDTA,静置5min;加入15μl无水乙醇,漩涡混匀;3700rpm离心30min;倒置离心15sec,加入50ml70%乙醇,漩涡混匀;3700rpm离心15min;倒置离心15sec,置于95℃金属浴上;加入10μl CBL后进行变性5min,最后-20℃2min上测序仪(ABI3730)测序。
(7)结果判断:分别将测序结果与ABCB4参考序列(GeneBank序列编号:NC_000007.14)进行比对,根据实际突变情况对结果进行报告。
实施例3
取20例临床外周血样品,按实施例2所述方法提取基因组、配制试剂并检测。每份样品加入检测体系PCR反应液中2μl。同时做阳性对照、阴性对照和空白对照各一份。PCR扩增后,20例样本的琼脂糖凝胶电泳结果如图1所示。进一步进行测序后发现在20例样本中,只有一个样本未发生突变,其余样本全部发生ABCB4 c.3508-16T>C位点突变,该位点突变测序演示见图2。
序列表
<110> 济南艾迪康医学检验中心有限公司
<120> 检测ABCB4 c.3508-16T>C位点突变的引物和方法
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
tgtaaaacga cggccagttg taaatagaac tgtcaactgt taagc 45
<210> 2
<211> 39
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
aacagctatg accatgtttt catggttgac agcaaaatc 39
<210> 3
<211> 18
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
tgtaaaacga cggccagt 18
<210> 4
<211> 16
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
aacagctatg accatg 16
Claims (7)
1.检测ABCB4 c.3508-16T>C位点突变情况的引物,其特征在于,包括:
扩增ABCB4 c.3508-16T>C位点的引物,其碱基序列为:
ABCB4-A-F:
TGTAAAACGACGGCCAGTTGTAAATAGAACTGTCAACTGTTAAGC
ABCB4-A-R:AACAGCTATGACCATGTTTTCATGGTTGACAGCAAAATC。
2.如权利要求1所述的引物,其特征在于,还包括测序引物,其碱基序列为:
M13F:TGTAAAACGACGGCCAGT
M13R:AACAGCTATGACCATG。
3.如权利要求1所述的引物,其特征在于,ABCB4-A-F和ABCB4-A-R的摩尔浓度比1:1。
4.如权利要求1所述的引物,其特征在于,M13F和M13R的摩尔浓度比1:1。
5.检测ABCB4 c.3508-16T>C位点突变情况的方法,包括以下步骤:
(1)提取血液基因组DNA;
(2)利用一对PCR扩增引物扩增步骤(1)中提取的DNA,获取扩增产物;
(3)利用一对测序引物对步骤(2)中的扩增产物进行测序;
(4)对测序结果进行判断,确定ABCB4 c.3508-16T>C位点是否发生突变;
其中所述一对PCR扩增引物的碱基序列为:
ABCB4-A-F:
TGTAAAACGACGGCCAGTTGTAAATAGAACTGTCAACTGTTAAGC
ABCB4-A-R:AACAGCTATGACCATGTTTTCATGGTTGACAGCAAAATC;
所述一对测序引物的碱基序列为:
M13F:TGTAAAACGACGGCCAGT
M13R:AACAGCTATGACCATG。
6.一种检测ABCB4 c.3508-16T>C位点突变的试剂盒,其特征在于,所述试剂盒包括:
(i)血液基因组DNA抽提试剂;
(ii)检测体系PCR扩增反应液:包括扩增ABCB4 c.3508-16T>C位点的引物,其碱基序列为:
ABCB4-A-F:
TGTAAAACGACGGCCAGTTGTAAATAGAACTGTCAACTGTTAAGC
ABCB4-A-R:AACAGCTATGACCATGTTTTCATGGTTGACAGCAAAATC
(iii)阳性对照品和阴性对照品。
7.如权利要求6所述的试剂盒,其特征在于,所述试剂盒还包括测序体系试剂:
包括测序引物,其碱基序列为:
M13F:TGTAAAACGACGGCCAGT
M13R:AACAGCTATGACCATG。
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