CN103882127B - 用于预测患2型糖尿病肾病发生风险的试剂盒 - Google Patents
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Abstract
本发明涉及一种用于预测患2型糖尿病肾病发生风险的试剂盒，该试剂盒包括标记物，该标记物为在人醌式二氢蝶呤还原酶基因rs3733570SNP位点的基因型，该基因型的存在标志患2型糖尿病肾病的发生风险，该试剂盒不但保证了一定的准确性及灵敏度，同时具有检测周期短，灵敏度高，操作简单，成本低等优点。
Description
技术领域
本发明涉及分子生物学和医学领域，特别涉及一种用于预测患2型糖尿病肾病发生风险的试剂盒。
背景技术
糖尿病是一种糖、蛋白质、脂肪代谢障碍的慢性综合征，继发于胰岛素分泌不足，或靶组织产生抗胰岛素性；有两种主要型：胰岛素依赖性糖尿病（1型）和非胰岛素依赖性糖尿病（2型）。
2型糖尿病（type2diabetesmellitus，T2DM）的发病受到环境和基因双重因素的影响。遗传因素能增加机体发生2型糖尿病的危险性。目前T2DM的发病率在所有种族和年龄段均逐渐上升。虽然进行了大量的基因组关联分析，但是大部分T2DM相关基因仍然未知。
2型糖尿病肾病（diabeticnephropathy，DN）是T2DM的一个常见并发症，其发展也受到环境和基因双重因素的影响；DN能导致T2DM患者终末期肾病；是成人慢性肾功能衰竭病首要原因；也是糖尿病患者发病和致死的主要原因。所以当务之急是研究DN的易感基因，以此来预测2型糖尿病肾病的发生风险及开展针对此基因的药物治疗；目前一些基因组扫描（genome-widescans，GWS）和相关性研究已经发现一些基因与DN密切相关，但是DN发生的所有易感基因并未完全明确。因此为了研究2型糖尿病肾病及其发病机制，预测2型糖尿病肾病的发生风险，需要生物学家及医学家更多的发现2型糖尿病肾病的相关基因。
发明内容
本发明的目的在于，通过检测rs3733570SNP（singlenucleotidepolymorphism）位点的基因型提供一种预测患2型糖尿病肾病发生风险的试剂盒。
本发明具体技术方案如下：
本发明提供一种用于预测患2型糖尿病肾病发生风险的试剂盒，该试剂盒包括标记物，该标记物为在人醌式二氢蝶呤还原酶基因上存在的rs3733570SNP位点的基因型，该基因型可以用来预测患2型糖尿病肾病的发生风险。
rs3733570的相关信息可从http://www.ncbi.nlm.nih.gov/SNP/snp_ref.cgi?rs=3733570中找到依据。
进一步的改进，所述rs3733570的T等位基因的存在标志着患有2型糖尿病肾病的发生风险增高。
进一步的改进，所述rs3733570的T/T基因型的存在标志着患有2型糖尿病肾病的发生风险增高。
进一步的改进，所述rs3733570共显性中C/T和T/T基因型的存在，或显性中C/T和T/T基因型的存在，或隐性中T/T基因型的存在标志着患有2型糖尿病肾病的发生风险增高。
进一步的改进，共显性中T/T基因型患2型糖尿病肾病的发生风险是C/C基因型的7-8倍；C/T基因型患2型糖尿病肾病的发生风险是C/C基因型的3-4倍。
进一步的改进，显性中C/T和T/T基因型患2型糖尿病肾病的发生风险是C/C基因型的4-5倍。
进一步的改进，隐性中T/T基因型患2型糖尿病肾病的发生风险是C/C和C/T基因型的3-4倍。
进一步的改进，所述试剂盒还包括PCR扩增体系所用的正义引物，反义引物，PCR试剂和PCR反应液；其中，正义引物为TTGCCAGTGTATAGGTAAGG，反义引物为ATTCATTCCAGTGTAGA，PCR试剂为2×ESTaqMasterMix；PCR反应液为：抗凝全血3%，0.5μM正义引物，0.5μM反义引物，2×ESTaqMasterMix50%。
本发明另一方面还提供用于预测患2型糖尿病肾病发生风险的试剂盒的使用方法，该使用方法包括如下步骤：
a.收取EDTA-K2抗凝静脉全血；
b.通过全血PCR扩增体系扩增rs3733570所在外显子片段；
c.将b步骤获得的PCR产物纯化；
d.将步骤c得到的纯化产物，经测序仪（3730xl，ABI）测序；
e.分析结果。
进一步的改进，所述扩增步骤为：所述扩增步骤为：94℃3分钟，55℃3分钟循环4次，94℃变性5分钟后进入循环，94℃变性30秒，52℃退火30秒，72℃延伸90秒，40个周期后，72℃延伸5分钟，4℃保持。
本发明的有益效果：本发明所提供的试剂盒用于检测醌式二氢蝶呤还原酶基因rs3733570SNP位点的基因型，该可以用来预测患2型糖尿病肾病的发生风险；其可通过特异性引物扩增目的区域，分析鉴定该SNP的基因型；该试剂盒不但保证了一定的准确性及灵敏度，同时具有检测周期短，灵敏度高，操作简单，成本低等优点。
具体实施方式
实施例1
用于预测患2型糖尿病肾病发生风险的试剂盒，包括标记物，PCR扩增体系所用的正义引物，反义引物，PCR试剂和PCR反应液；其中，GAI标记物为在人醌式二氢蝶呤还原酶rs3733570SNP位点的基因型；正义引物为TTGCCAGTGTATAGGTAAGG，反义引物为ATTCATTCCAGTGTAGA，PCR试剂为2×ESTaqMasterMix；PCR反应液为：抗凝全血3%，0.5μM正义引物，0.5μM反义引物，2×ESTaqMasterMix50%。
所述试剂盒的使用方法包括如下步骤：
a.收取EDTA-K2抗凝静脉全血；
b.通过全血PCR扩增体系进行扩增：在94℃3分钟，55℃3分钟循环4次，94℃变性5分钟后进入循环，94℃变性30秒，52℃退火30秒，72℃延伸90秒，40个周期后，72℃延伸5分钟，4℃保持；
c.将b步骤获得的PCR产物纯化；
d.将步骤c得到的纯化产物，经测序仪（3730xl，ABI）测序；
e.分析结果。
试验例1
利用实施例1的试剂盒，按照实施例1公开的方法检测的多态性，发现rs3733570的基因型在2型糖尿病肾病的患者与正常患者对照分布不同。
1.研究对象
所选取的研究对象为河北省唐山市开滦医院入院病人；研究对象分为高龄未患糖尿病对照组（NC），2型糖尿病组（T2DM），2型糖尿病肾病组（DN）三组，NC入组标准为：年龄≥60岁，血糖水平正常（空腹血糖＜6.1mmol/L而且餐后2小时血糖＜7.8mmol/L），无既往糖尿病史；T2DM标准为符合1999年WHO糖尿病诊断标准并排除具有T1DM及年轻的成人起病糖尿病（MODY）等特殊类型糖尿病；另外需无糖尿病肾病的病史，尿常规蛋白阴性，血肌酐水平正常；DN入组标准为除前述糖尿病的诊断标准外应同时具备，眼科诊断的糖尿病性视网膜病变，尿蛋白阳性（至少2次尿常规蛋白阳性（+～3+）或24小时尿蛋白定量≥0.5g）；无血尿：尿常规红细胞＜3个/高倍视野；各组入组患者必须同时排除：①临床诊断的除糖尿病性肾病以往的其他原发性或继发性肾脏疾病；②血液系统疾病；③恶性肿瘤；本研究的注册号：ChiCTR-TNRC-11001489；伦理委员会批件文号为2006医伦字5号。
2.检测方法：
rs3733570位点所在DNA的扩增
所用试剂2×ESTaqMasterMix（康为世纪，CW0690）
所用引物
senseTTGCCAGTGTATAGGTAAGG
antisenseATTCATTCCAGTGTAGA
所有入组病人留取2mlEDTA-K2抗凝静脉全血分装后保存于-80℃，在94℃3分钟，55℃3分钟循环4次以破坏细胞膜及核膜释放基因组DNA；通过PCR扩增体系进行扩增；PCR扩增按如下程序进行：94℃变性5分钟后进入循环，94℃变性30秒，52℃退火30秒，72℃延伸90秒，40个周期后72℃延伸5分钟，4℃保持；PCR产物经纯化后经测序仪（3730xl，ABI）分析后获得序列；获得的序列观察rs3733570为何种核苷酸；
3.统计学分析方法
描述性统计资料年龄用方差分析，性别用卡方检验；基因型分布温-哈平衡（Hardy-Weinbergequilibrium，HWE）用HW计算器；运用非条件logistic回归计算相对危险度(oddsratio，OR)和95%可信区间（95%confidenceinterval，95%CI)，p<0.05被认为有统计学差异。
4.结果
研究对象的临床特征见表1；
rs3733570在人群中的分布见表2；
rs3733570与2型糖尿病肾病的相关性见表3；
表一研究对象的临床特征
Pa未患糖尿病对照组与各组相比。
从表1可看出，T2DM和DN组与正常人相比年龄有统计学差异（p<0.001)，T2DM和DN组相比年龄无统计学差异，三组性别无统计学差异(p>0.05)。
表二rs3733570在人群中的分布
从表2中可看出，三组基因分布符合HWE模型，结果可靠。
表三rs3733570与2型糖尿病肾病的相关性
a未患糖尿病对照组与2型糖尿病肾病组相比。
从表3中可看出，
在共显性模型中T/T基因型患2型糖尿病肾病的发生风险是C/C基因型的7.69倍；C/T基因型患2型糖尿病肾病的发生风险是C/C基因型的3.67倍；
在显性模型中C/T和T/T基因型患2型糖尿病肾病的发生风险是C/C基因型的4.47倍；
在隐性模型中T/T基因型患2型糖尿病肾病的发生风险是C/C和C/T基因型的3.08倍。
5.结论
由以上表中可以看出，rs3733570与糖尿病和2型糖尿病肾病均具有相关性，并且rs3733570与2型糖尿病肾病相关有统计学差异。
Claims (8)
1.一种用于预测患2型糖尿病肾病发生风险的试剂盒，其特征在于，所述试剂盒包括标记物，所述标记物为在人醌式二氢蝶呤还原酶基因上存在的rs3733570SNP位点的基因型，所述基因型可以用来预测患2型糖尿病肾病的发生风险。
2.如权利要求1所述的用于预测患2型糖尿病肾病发生风险的试剂盒，其特征在于，所述rs3733570的T等位基因的存在标志着患有2型糖尿病肾病的发生风险增高。
3.如权利要求1所述的用于预测患2型糖尿病肾病发生风险的试剂盒，其特征在于，所述rs3733570的T/T基因型的存在标志着患有2型糖尿病肾病的发生风险增高。
4.如权利要求1所述的用于预测患2型糖尿病肾病发生风险的试剂盒，其特征在于，所述rs3733570共显性中C/T和T/T基因型的存在，或显性中C/T和T/T基因型的存在，或隐性中T/T基因型的存在标志着患有2型糖尿病肾病的发生风险增高。
5.如权利要求4所述的用于预测患2型糖尿病肾病发生风险的试剂盒，其特征在于，共显性中T/T基因型患2型糖尿病肾病的发生风险是C/C基因型的7-8倍；C/T基因型患2型糖尿病肾病的发生风险是C/C基因型的3-4倍。
6.如权利要求4所述的用于预测患2型糖尿病肾病发生风险的试剂盒，其特征在于，显性中C/T和T/T基因型患2型糖尿病肾病的发生风险是C/C基因型的4-5倍。
7.如权利要求4所述的用于预测患2型糖尿病肾病发生风险的试剂盒，其特征在于，隐性中T/T基因型患2型糖尿病肾病的发生风险是C/C和C/T基因型的3-4倍。
8.如权利要求1所述的用于预测患2型糖尿病肾病发生风险的试剂盒，其特征在于，所述试剂盒还包括PCR扩增体系所用的正义引物，反义引物，PCR试剂和PCR反应液；所述正义引物为TTGCCAGTGTATAGGTAAGG，所述反义引物为ATTCATTCCAGTGTAGA，所述PCR试剂为2×ESTaqMasterMix；所述PCR反应液为：抗凝全血3％，0.5μM正义引物，0.5μM反义引物，2×ESTaqMasterMix50％。
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| CN103981280B (zh) * | 2014-03-13 | 2016-08-17 | 河北联合大学 | 用于预测患2型糖尿病肾病发生风险的标记物及其用途 |
| CN109765377A (zh) * | 2018-08-17 | 2019-05-17 | 南方医科大学 | 用于诊断糖尿病肾病早期阶段的尿EVs生物标志物 |
| CN111560430B (zh) * | 2020-06-17 | 2023-05-23 | 中南大学湘雅二医院 | 检测rs1766位点多态性的试剂及其应用 |
| CN112526132A (zh) * | 2020-11-23 | 2021-03-19 | 华中科技大学 | 预测2型糖尿病患者出现记忆力衰退的风险的方法 |
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| CN1548557A (zh) * | 2003-05-21 | 2004-11-24 | 中国医学科学院基础医学研究所 | 用于预测2型糖尿病易感性的试剂盒及引物 |
| CN101230392A (zh) * | 2007-12-06 | 2008-07-30 | 卫生部北京医院 | 一种预测2型糖尿病易感性的方法及试剂 |
| CN101676404A (zh) * | 2008-09-18 | 2010-03-24 | 卫生部北京医院 | 一种线粒体nd2基因snp预测2型糖尿病易感性的方法及试剂 |
| CN103154272A (zh) * | 2010-08-25 | 2013-06-12 | 香港中文大学 | 利用遗传标志物和阵列预测糖尿病相关并发症风险的方法和试剂盒 |
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| JP2008522597A (ja) * | 2004-12-13 | 2008-07-03 | エフ．ホフマン−ラ ロシュ アーゲー | Ｉｉ型糖尿病に関連する単一ヌクレオチド多型（ｓｎｐ） |
| KR101374304B1 (ko) * | 2005-06-20 | 2014-03-14 | 디코드 제네틱스 이에이치에프 | 타입 2 당뇨병의 위험에 대한 진단 마커인 ｔｃｆ7ｌ2유전자의 유전적 변이체 |
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| CN101230392A (zh) * | 2007-12-06 | 2008-07-30 | 卫生部北京医院 | 一种预测2型糖尿病易感性的方法及试剂 |
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| CN103154272A (zh) * | 2010-08-25 | 2013-06-12 | 香港中文大学 | 利用遗传标志物和阵列预测糖尿病相关并发症风险的方法和试剂盒 |
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