CN108330179A - 一种TNF-α拮抗剂应答疗效SNP位点检测试剂盒 - Google Patents
一种TNF-α拮抗剂应答疗效SNP位点检测试剂盒 Download PDFInfo
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Abstract
一种TNF‑α拮抗剂应答疗效SNP位点检测试剂盒,本发明涉及分子生物学和医学领域，本试剂盒针对人类基因组DNA中肿瘤坏死因子拮抗剂应答效应预测的5个SNP位点设计特异性引物和探针，与携带相应报告探针的agPlex‑TAG磁珠进行杂交反应，通过显色反应在luminex 200仪器上进行检测。通过对信号值的读取判读各个SNP位点的碱基型别进行判定。本发明的有益效果在于：本发明针对类风湿关节炎、强直性脊柱炎、银屑病等患者使用肿瘤坏死因子拮抗剂的有效性相关基因位点设计特异引物和探针以及相应报告探针，可分型检测5个SNP位点的型别；检测试剂盒可以灵敏、快速低检测人全血样本中5个SNP型别，结合多个SNP位点可对患者使用肿瘤坏死因子拮抗剂治疗有效性进行预测。
Description
技术领域
本发明涉及分子生物学和医学领域，更具体的，本发明涉及一组肿瘤坏死因子拮抗剂应疗效SNP位点检测的试剂盒。通过同时检测肿瘤坏死因子拮抗剂应答疗效密切相关的TNF-a基因、TNFRSF1B基因和FCGR3A基因的单核苷酸多态性位点(SNP)基因型来评估肿瘤坏死因子拮抗剂在克罗恩病、强直性脊柱炎，银屑病关节炎等炎症性疾病的应答效应。
背景技术
肿瘤坏死因子α是风湿性疾病，尤其是类风湿关节炎发病机制中最重要的细胞因子之一，其主要的生物学作用包括：导致关节炎症和软骨破坏，诱导其他炎性细胞因子的释放，介导感染和败血症，参与肿瘤监视等。肿瘤坏死因子α拮抗剂可以通过与肿瘤坏死因子α特异性结合而阻断肿瘤坏死因子α生物活性的发挥，从而达到控制炎症、持续缓解病情的目的。
肿瘤坏死因子(TNF)-α拮抗剂已成为治疗风湿性疾病最有力的武器之一。目前临床使用的TNF-α拮抗剂主要有依那西普、阿达木单抗和英夫利昔单抗，它们均可缓解及阻止类风湿关节炎的临床及影像学进展，显著减轻类风湿关节炎患者的症状、改善功能及提高生活质量等；可显著改善银屑病性关节炎患者的关节炎症状，减缓其影像学进展。可降低强直性脊柱炎患者的疾病活动度，延缓影像学进展以及改善生活质量。长期随访研究显示TNF-α拮抗剂用于类风湿关节炎，强直性脊柱炎和银屑病关节炎治疗安全有效。
肿瘤坏死因子(TNF)-α拮抗剂费用昂贵，部分患者初次治疗，临床疗效不佳甚至无反应，而且接受治疗的患者有出现结核、真菌、细菌感染等并发症的风险，寻找能预测AS患者对TNF-α拮抗剂治疗有效性的标志物显得十分重要。
多项研究及Meta分析提示：TNF-α-308G(rs1800629),TNF-α-857C(rs1799724),TNF-a 238G(rs361525),TNFRSF1B 676T(rs1061622),FCGR3A449G(rs396991)与使用肿瘤坏死因子抑制剂治疗应答相关。
TNF-α启动子-308启动子G/A位点和AS有关。-308位点GA+AA基因型与AS骶髂关节炎严重程度可能存在关联。有研究表明使用TNF-α拮抗剂24周后，TNF-a-308G/G纯合子的AS患者BASDAI改善优于A/A、A/G的患者。TNF-α拮抗剂有效的患者中-308A基因频率为22％，而无效的患者中-308A基因频率为37％。
已有研究表明rs1799724(-857C/T)单核苷酸多态性与AS有显着关联,-857T是中国南方人群AS发病的危险因素。-857TT纯合子较CT杂合子对TNF-a拮抗剂6周治疗后的AS病人的炎症指标、BASFI改善更显著。在治疗类风湿关节炎患者中，使用TNF-a抑制剂(依那西普)治疗后，TNF-308AA基因型对其应答反应明显较TNF-308GG基因型。TNF-238GA基因型患者对英夫利昔单抗的应答较GG基因型差。
TNF受体超家族基因，TNFRSF1B676T>G(rs1064622)，主要的功能是影响细胞凋亡。IgGFc段受体基因FCGR3A 449G>T(rs396991)影响与人IgG连接的亲和力。这两个基因SNP位点也与TNF-a拮抗剂治疗RA疗效相关。TNFRSF1B676T>G(rs1064622)GG基因型使用TNF-a拮抗剂治疗效应差；银屑病患者写到FCGR3A449G>T(rs396991)AA基因型的治疗应答较AC和CC基因型弱。
发明内容
本试剂盒针对人类基因组DNA中肿瘤坏死因子拮抗剂应答效应预测的5个SNP位点，即TNF-a基因(308G>A)，TNF-a(238G>A)，TNF-a(857C>T)，TNFRSF1B(676T>G)，FCGR3A(175A>C)。设计特异性引物和探针，与携带相应报告探针的MagPlex-TAG磁珠进行杂交反应，通过显色反应在luminex 200仪器上进行检测。通过对信号值的读取判读各个SNP位点的碱基型别进行判定。
试剂盒中包含的特异引物序列如下：
试剂盒中包含的特异探针序列如下：
试剂盒中包含的报告探针序列如下：
一种TNF-α拮抗剂应答疗效SNP位点检测的方法，所述的方法包括如下的步骤：首先进行PCR反应，实现扩增；再进行多重OLA反应，标记和连接；然后进行杂交反应，最后进行基因型的分析。
进一步的，PCR反应体系如下：2x qiagen Hotstar MM 5ul,primer mix 1ul，DNA样本2ul,灭菌水2ul；PCR反应条件为95℃、15min，进行30个循环的94℃,30秒，60℃,30秒，72℃,30秒；72℃、7分钟，4℃维持。
进一步的，多重OLA反应如下：配制2xOLA master mix，将OLA master mix与PCR反应产物混合，混匀后进行连接反应。
进一步的，配制2xOLA master mix包括有：10x Taq Ligase buffer 2ul,40000U/ml的Taq DNA Ligase 0.25ul，野生型探针mix 1ul，突变型探针mix2ul，去离子水4.75ul。
进一步的，连接反应条件如下：96℃2min,30个循环的94℃15s,37℃1min；4℃维持。
进一步的，杂交反应的过程如下：选择相应的报告探针的磁珠，重悬，然后将每种磁珠混合，稀释最多至100u颗/ul，用2X Tm hybridization buffer，混合后加入磁珠混合物至每孔中，添加1-5ul OLA reaction和25ul dH2O至每个孔，进行PCR反应：96℃90s,37℃30min，吸走上清，用1x Tm hybridization buffer重悬磁珠，吸磁30-60s，再次吸走上清，重复用1x Tm hybridization buffer重悬磁珠，吸磁30-60s，第三次吸走上清，用1x Tmhybridization buffer重悬磁珠，在37℃温育15min，加入50ul反应产物至LUMINEX中分析。
或者，
杂交反应的过程如下：1、选择磁珠，并重悬；2、将每种磁珠混合，并稀释至100u颗/ul，用2X Tm hybridization buffer，振荡混合；3、加入磁珠混合物至每孔中；4、添加样品至每孔中；5、进行PCR反应：96℃90s，37℃30min；6、准备6ug/ml SAPE在1x hybridizationbuffer；7、添加100ul SAPE mix，混匀；8、37℃温育15min；9、加入100ul至37℃的luminex中分析。
本发明的有益效果在于：本发明针对类风湿关节炎、强直性脊柱炎、银屑病等患者使用肿瘤坏死因子拮抗剂的有效性(反应应答)相关基因位点设计特异引物和探针以及相应报告探针，可分型检测5个SNP位点的型别。本发明提供的检测试剂盒可以灵敏、快速低检测人全血样本中5个SNP型别，结合多个SNP位点可对患者使用肿瘤坏死因子拮抗剂治疗有效性进行预测。指导临床治疗选择。
具体实施方式
实施例1
本发明提供一种试剂盒，试剂盒中包括的特异引物序列如下：
试剂盒中包括的特异探针序列如下：
试剂盒中包括的报告探针序列如下：
实施例2
5个SNP位点在同一管进行PCR反应，反应的体系为总体积10μl，包含2x qiagenHotstar MM 5ul,primer mix 1ul，DNA样本2ul,灭菌水2ul。
在ABI9700型PCR扩增仪上进行反应，反应条件为95℃、15min，进行30个循环的94℃,30秒，60℃,30秒，72℃,30秒；72℃、7分钟，4℃维持。
多重OLA反应：配制2xOLA master mix：10x Taq Ligase buffer 2ul,Taq DNALigase(40,000U/ml)0.25ul，野生型探针mix(100nM each)1ul,突变型探针mix(2.5uMeach)2ul，去离子水4.75ul。将OLA master mix与反应产物混合：2xOLA master mix 10ul,扩增的PCR产物5ul,灭菌去离子水5ul。上下吹打混匀，盖上反应管，进行在ABI9700型PCR扩增仪上连接反应。96℃2min,30个循环的94℃15s,37℃1min；4℃维持。
杂交反应-洗涤程序：选择相应报告探针的MagPlex-TAG磁珠，并重悬，将每种磁珠混合，并稀释至100u颗/ul，用2X Tm hybridization buffer，振荡混合20s；加入25ul磁珠混合物至每孔中(应该提供2500颗珠子/每种反应)。添加1-5ul OLA reaction和25ul dH2O至每个孔，调整H2O的体积，使总体积接近50ul。盖上盖子，进行PCR反应：96℃90s，37℃30min。放于磁力板中30s-60s，使磁珠吸住，小心吸走上清，别吸走磁珠；用1x Tmhybridization buffer 75ul，重悬MagPlex-TAG磁珠，吸磁30-60s，小心吸走上清，别吸走磁珠；重复用1x Tm hybridization buffer 75ul，重悬MagPlex-TAG磁珠，吸磁30-60s，小心吸走上清；用75ul 1x Tm hybridization buffer(包含2-8ug/ml SAPE)，重悬磁珠，在37℃温育15min。在37℃中，加入50ul反应产物至LUMINEX中分析。
或者，
杂交反应-不洗涤程序：1.选择合适的MagPlex-TAG磁珠，并重悬；2.将每种磁珠混合，并稀释至100u颗/ul，用2X Tm hybridization buffer，振荡混合20s；3.加入20ul磁珠混合物至每孔中(应该提供2500颗珠子/每种反应)。4.添加5uL样品至每孔中；5.关上盖子，进行PCR反应，PCR反应：96℃90s，37℃30min；6.准备6ug/ml SAPE在1x hybridizationbuffer；7.添加100ul SAPE mix，轻柔混匀；8.37℃温育15min；9.加入100ul至37℃的luminex中分析。
结果分析
通过质粒及水对照，获得背景信号，检测样本结果时，减去背景后，数值大于200即为阳性反应。
检测报告中会提供5个SNP型别结果，检测TNF-a(308G>A)rs1800629，GG和AG基因型对肿瘤坏死因子拮抗剂治疗效果好；TNF-a(238G>A)rs361525，GG基因型使用英夫利昔单抗和依那西普治疗应答较好，而使用阿达木单抗的治疗应答较差；TNF-a(857C>T)rs1799724，CC基因型对英夫利昔单抗治疗应答较好，对依那西普和阿达木单抗应答较差；FCGR3A(175A>C)，rs396991，银屑病患者AA基因型治疗效果较差；TNFRSF1B(676T>G)rs1061622，GG基因型治疗效果较差。
TNF-a基因(308G>A)rs1800629，突变型对应的结果显示为AA,杂合型为AG,野生型为GG；
TNF-a基因(238G>A)rs361525，突变型对应的结果显示为AA,杂合型为AG,野生型为GG；
TNF-a基因(857C>T)rs1799724，突变型对应的结果显示为TT,杂合型为CT,野生型为CC；
FCGR3A基因(175A>C)rs396991，突变型对应的结果显示为CC,杂合型为AC,野生型为AA；
TNFRSF1B基因(676T>G)rs1061622，突变型对应的结果显示为GG,杂合型为TG,野生型为TT。
序列表
<110> 和康医疗
<120> 一种TNF-α拮抗剂应答疗效SNP位点检测试剂盒
<130> PJ1710735.06
<160> 25
<170> SIPOSequenceListing 1.0
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<211> 18
<212> DNA
<213> 人工序列("人工序列")
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gcctcaggac tcaacaca 18
<210> 2
<211> 20
<212> DNA
<213> 人工序列("人工序列")
<400> 2
acacaagcat caaggatacc 20
<210> 3
<211> 21
<212> DNA
<213> 人工序列("人工序列")
<400> 3
ctgtctggaa gttagaagga a 21
<210> 4
<211> 18
<212> DNA
<213> 人工序列("人工序列")
<400> 4
atctggagga agcggtag 18
<210> 5
<211> 21
<212> DNA
<213> 人工序列("人工序列")
<400> 5
gaagctgaga agatgaagga a 21
<210> 6
<211> 20
<212> DNA
<213> 人工序列("人工序列")
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ggagactcat aatgctggtt 20
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<211> 21
<212> DNA
<213> 人工序列("人工序列")
<400> 7
acacatcgtc actctcctat c 21
<210> 8
<211> 20
<212> DNA
<213> 人工序列("人工序列")
<400> 8
agacttcttg cgacttacca 20
<210> 9
<211> 19
<212> DNA
<213> 人工序列("人工序列")
<400> 9
ggtgttcaag gaggaagac 19
<210> 10
<211> 20
<212> DNA
<213> 人工序列("人工序列")
<400> 10
gtgcgtgtaa gaatcaggaa 20
<210> 11
<211> 45
<212> DNA
<213> 人工序列("人工序列")
<400> 11
tactacttct ataactcact taaaaatagg ttttgagggg catgg 45
<210> 12
<211> 45
<212> DNA
<213> 人工序列("人工序列")
<400> 12
cttaacattt aacttctata acacaatagg ttttgagggg catga 45
<210> 13
<211> 42
<212> DNA
<213> 人工序列("人工序列")
<400> 13
attaaacaac tcttaactac acaaagaccc ccctcggaat cg 42
<210> 14
<211> 42
<212> DNA
<213> 人工序列("人工序列")
<400> 14
tacaacatct cattaacata tacaagaccc ccctcggaat ca 42
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<212> DNA
<213> 人工序列("人工序列")
<400> 15
tacttcttta ctacaattta caactgggga ccccccctta ac 42
<210> 16
<211> 42
<212> DNA
<213> 人工序列("人工序列")
<400> 16
acacttatct ttcaattcaa ttactgggga ccccccctta at 42
<210> 17
<211> 42
<212> DNA
<213> 人工序列("人工序列")
<400> 17
cataatcaat ttcaactttc tactccctgg gaatgcaagc at 42
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<213> 人工序列("人工序列")
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<212> DNA
<213> 人工序列("人工序列")
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aactttctct ctctattctt attttacttc tgcagggggc ttt 43
<210> 20
<211> 43
<212> DNA
<213> 人工序列("人工序列")
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cactacacat ttatcataac aaattacttc tgcagggggc ttg 43
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<212> DNA
<213> 人工序列("人工序列")
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ggacggggtt cagcct 16
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<213> 人工序列("人工序列")
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gagcagggag gatggg 16
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<213> 人工序列("人工序列")
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<213> 人工序列("人工序列")
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ggatgcagtc tgcacg 16
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Claims (10)
1.一种TNF-α拮抗剂应答疗效SNP位点检测试剂盒，其特征在于，所述的试剂盒包括有至少一对如下序列的引物：
2.如权利要求1所述的一种TNF-α拮抗剂应答疗效SNP位点检测试剂盒，其特征在于，所述的试剂盒还包括特异探针，所述的特异探针序列如下：
3.如权利要求1所述的一种TNF-α拮抗剂应答疗效SNP位点检测试剂盒，其特征在于，所述的试剂盒还包括报告探针，所述的报告探针序列如下：
4.一种TNF-α拮抗剂应答疗效SNP位点检测的方法，其特征在于，所述的方法包括如下的步骤：首先进行PCR反应，实现扩增；再进行多重OLA反应，标记和连接；然后进行杂交反应，最后进行基因型的分析。
5.如权利要求4所述的一种TNF-α拮抗剂应答疗效SNP位点检测的方法，其特征在于，PCR反应体系如下：2x qiagen Hotstar MM 5ul,primer mix 1ul，DNA样本2ul,灭菌水2ul；PCR反应条件为95℃、15min，进行30个循环的94℃,30秒，60℃,30秒，72℃,30秒；72℃、7分钟，4℃维持。
6.如权利要求4所述的一种TNF-α拮抗剂应答疗效SNP位点检测的方法，其特征在于，多重OLA反应如下：配制2xOLA master mix，将OLA master mix与PCR反应产物混合，混匀后进行连接反应。
7.如权利要求6所述的一种TNF-α拮抗剂应答疗效SNP位点检测的方法，其特征在于，配制2xOLA master mix包括有：10x Taq Ligase buffer 2ul,40000U/ml的Taq DNA Ligase0.25ul，野生型探针mix 1ul，突变型探针mix 2ul，去离子水4.75ul。
8.如权利要求6所述的一种TNF-α拮抗剂应答疗效SNP位点检测的方法，其特征在于，连接反应条件如下：96℃2min,30个循环的94℃15s,37℃1min；4℃维持。
9.如权利要求4所述的一种TNF-α拮抗剂应答疗效SNP位点检测的方法，其特征在于，杂交反应的洗涤过程如下：选择相应的报告探针的磁珠，重悬，然后将每种磁珠混合，稀释最多至100u颗/ul，用2X Tm hybridization buffer，混合后加入磁珠混合物至每孔中，添加1-5ul OLA reaction和25ul dH2O至每个孔，进行PCR反应：96℃90s,37℃30min，吸走上清，用1x Tm hybridization buffer重悬磁珠，吸磁30-60s，再次吸走上清，重复用1x Tmhybridization buffer重悬磁珠，吸磁30-60s，第三次吸走上清，用1x Tm hybridizationbuffer重悬磁珠，在37℃温育15min，加入50ul反应产物至LUMINEX中分析。
10.如权利要求4所述的一种TNF-α拮抗剂应答疗效SNP位点检测的方法，其特征在于，杂交反应的不洗涤程序过程如下：1、选择磁珠，并重悬；2、将每种磁珠混合，并稀释至100u颗/ul，用2X Tm hybridization buffer，振荡混合；3、加入磁珠混合物至每孔中；4、添加样品至每孔中；5、进行PCR反应：96℃90s，37℃30min；6、准备6ug/ml SAPE在1xhybridization buffer；7、添加100ul SAPE mix，混匀；8、37℃温育15min；9、加入100ul至37℃的luminex中分析。
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| CN113388678A (zh) * | 2021-06-30 | 2021-09-14 | 南通市第一人民医院 | 一种强直性脊柱炎易感基因的检测引物和探针及检测方法 |
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