CN110305958A - 一种msi检测方法 - Google Patents
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Abstract
本发明公开了一种MSI检测方法,包括如下步骤:S1、分别提取同一患者的正常粘膜组织的DNA与肿瘤组织的DNA,并分别选取贝斯塔遗传标记:Bat26、Bat25、D5S346、D2S123和D17S250这5个位点进行扩增引物标记荧光;S2、PCR扩增;S3、将步骤S2中得到的PCR扩增产物进行毛细管电泳。本发明采用多重PCR单管扩增,结合优化反应条件及进行标记荧光,提高分析质量和通量,节省试剂跟耗材,降低成本。
Description
技术领域
本发明涉及生物检测技术领域,具体涉及一种MSI检测方法。
背景技术
MSI是指与正常组织DNA相比,在肿瘤DNA中某一微卫星由于复制错误引起重复单位的插入或缺失而造成的微卫星长度的任何改变,从而出现新的微卫星等位基因现象。MSI检测在结直肠癌中主要用于判断预后,预测5-FU药物疗效,帮助筛查林奇综合征。传统检测方法中,主要是针对Bat25、Bat26、D2S123、D17S250、D5S346五个位点单独扩增,通量较低。
发明内容
针对现有技术的不足,本发明旨在提供一种MSI检测方法,采用多重PCR单管扩增,结合优化反应条件及进行标记荧光,提高分析质量和通量,节省试剂跟耗材,降低成本。
为了实现上述目的,本发明采用如下技术方案:
一种MSI检测方法,包括如下步骤:
S1、分别提取同一患者的正常粘膜组织的DNA与肿瘤组织的DNA,并分别选取贝斯塔遗传标记:Bat26,Bat25,D5S346,D2S123和 D17S250这5个位点进行扩增引物标记荧光;
S2、分别对步骤S1中得到的同一患者的正常粘膜组织的DNA与肿瘤组织的DNA进行PCR扩增:
S2.1、取离心管,加入12μl的DNA和0.5μl的DNA Polymerase 震荡混匀,离心;
S2.2、在装有PCR预混液的8联管中的前5个孔中分别加入2μl 步骤S2.1中离心后的混合液;
S2.3、盖紧管盖,震荡混匀,离心,进行PCR扩增;扩增条件为:
1)95℃,3min;
2)95℃,30秒,52℃,30秒,72℃,60秒;35个循环;
3)72℃,10min。
S3、分别对步骤S2中得到的同一患者的正常粘膜组织的DNA与肿瘤组织的DNA的PCR扩增产物在基因分析仪上按照如下过程进行毛细管电泳:
分别步骤S2.3中得到的8联管的前五个孔中的PCR扩增产物各 Iμl加入到新的离心管中,然后向所述新的离心管中加入0.5ul LIZ500和10ul HIDI,震荡混匀,离心,在基因分析仪上进行毛细管电泳,得到检测结果。
进一步地,所述扩增引物的序列如下:
针对Bat25的扩增引物为:
BAT-25F:FAM-ATGGAGGATGACGAGTTG;
BAT-25R:CAGGCTTGTACTTTACAGC;
针对Bat26的扩增引物为:
BAT-26F:HEX-ATTGGATATTGCAGCAGTCA;
BAT-26R:GCTCCTTTATAAGCTTCTTCAG;
针对D2S123的扩增引物为:
D2S123F:FAM-AAACAGGATGCCTGCCTTTA;
D2S123R:CTGACTTGGATACCATCTATCT;
针对D17S250的扩增引物为:
D17S250F:HEX-CAGGCTGGTCTCAAACTC;
D17S250R:GGAAGAATCAAATAGACAAT;
针对D5S346的扩增引物为:
D5S346F:TMR-TGGTTTCCATTGTAGCATCTTGAC;
D5S346R:CTGGTTGTTTCCGTAGTATATG。
本发明的有益效果在于:本发明采用多重PCR单管扩增,结合优化反应条件及进行标记荧光,提高分析质量和通量,节省试剂跟耗材,降低成本。
附图说明
图1为属于同一患者的正常组织DNA与肿瘤组织的DNA的毛细血管电泳检测结果中,Bat25、D2S123的对比情况示意图,其中图1(a) 为正常组织DNA的Bat25(左)与D2S123(右)的检测结果,图1(b) 为肿瘤组织DNA中Bat25(左)与D2S123(右)的检测结果。
图2为属于同一患者的正常组织DNA与肿瘤组织的DNA的毛细血管电泳检测结果中,Bat26、D17S250的对比情况示意图,其中图2 (a)为正常组织DNA的Bat26(左)与D17S250(右)的检测结果,图2(b)为肿瘤组织DNA中Bat26(左)与D17S250(右)的检测结果。
图3为属于同一患者的正常组织DNA与肿瘤组织的DNA的毛细血管电泳检测结果中,D5S346的对比情况示意图,其中图3(a)为正常组织DNA的D5S346的检测结果,图3(b)为肿瘤组织DNA中D5S346 的检测结果。
具体实施方式
以下将结合附图对本发明作进一步的描述,需要说明的是,本实施例以本技术方案为前提,给出了详细的实施方式和具体的操作过程,但本发明的保护范围并不限于本实施例。
本实施例提供一种MSI检测方法,包括如下步骤:
S1、分别提取同一患者的正常粘膜组织的DNA与肿瘤组织的DNA,并分别选取贝斯塔遗传标记(Bethesda Markers):Bat26,Bat25, D5S346,D2S123和D17S250这5个位点进行扩增引物标记荧光;
所述扩增引物的序列如下:
针对Bat25的扩增引物为:
BAT-25F:FAM-ATGGAGGATGACGAGTTG;
BAT-25R:CAGGCTTGTACTTTACAGC;
针对Bat26的扩增引物为:
BAT-26F:HEX-ATTGGATATTGCAGCAGTCA;
BAT-26R:GCTCCTTTATAAGCTTCTTCAG;
针对D2S123的扩增引物为:
D2S123F:FAM-AAACAGGATGCCTGCCTTTA;
D2S123R:CTGACTTGGATACCATCTATCT;
针对D17S250的扩增引物为:
D17S250F:HEX-CAGGCTGGTCTCAAACTC;
D17S250R:GGAAGAATCAAATAGACAAT;
针对D5S346的扩增引物为:
D5S346F:TMR-TGGTTTCCATTGTAGCATCTTGAC;
D5S346R:CTGGTTGTTTCCGTAGTATATG。
S2、分别对步骤S1中得到的同一患者的正常粘膜组织的DNA与肿瘤组织的DNA进行PCR扩增:
S2.1、取离心管,加入12μl的DNA和0.5μl的DNA Polymerase 震荡混匀,离心;
S2.2、在装有PCR预混液的8联管中的前5个孔中分别加入2μl 步骤S2.1中离心后的混合液;
S2.3、盖紧管盖,震荡混匀,离心,进行PCR扩增;扩增条件为:
1)95℃,3min;
2)95℃,30秒,52℃,30秒,72℃,60秒;35个循环;
3)72℃,10min。
S3、分别对步骤S2中得到的同一患者的正常粘膜组织的DNA与肿瘤组织的DNA的PCR扩增产物在基因分析仪上进行毛细管电泳;
具体过程为:
分别步骤S2.3中得到的8联管的前五个孔中的PCR扩增产物各 1μl加入到新的离心管中,然后向所述新的离心管中加入0.5ul LIZ500和10ul HIDI,震荡混匀,离心,在基因分析仪上进行毛细管电泳,得到检测结果。
图1为属于同一患者的正常组织DNA与肿瘤组织的DNA的毛细血管电泳检测结果中,Bat25、D2S123的对比情况示意图,其中图1(a) 为正常组织DNA的Bat25(左)与D2S123(右)的检测结果,图1(b) 为肿瘤组织DNA中Bat25(左)与D2S123(右)的检测结果。
图2为属于同一患者的正常组织DNA与肿瘤组织的DNA的毛细血管电泳检测结果中,Bat26、D17S250的对比情况示意图,其中图2 (a)为正常组织DNA的Bat26(左)与D17S250(右)的检测结果,图2(b)为肿瘤组织DNA中Bat26(左)与D17S250(右)的检测结果。
图3为属于同一患者的正常组织DNA与肿瘤组织的DNA的毛细血管电泳检测结果中,D5S346的对比情况示意图,其中图3(a)为正常组织DNA的D5S346的检测结果,图3(b)为肿瘤组织DNA中D5S346 的检测结果。
从图1-3中可以看出,本实施例方法的有效检测范围如表1所示。
表1
对于本领域的技术人员来说,可以根据以上的技术方案和构思,给出各种相应的改变和变形,而所有的这些改变和变形,都应该包括在本发明权利要求的保护范围之内。
Claims (2)
1.一种MSI检测方法,其特征在于,包括如下步骤:
S1、分别提取同一患者的正常粘膜组织的DNA与肿瘤组织的DNA,并分别选取贝斯塔遗传标记:Bat26,Bat25,D5S346,D2S123和D17S250这5个位点进行扩增引物标记荧光;
S2、分别对步骤S1中得到的同一患者的正常粘膜组织的DNA与肿瘤组织的DNA进行PCR扩增:
S2.1、取离心管,加入12μl的DNA和0.5μl的DNA Polymerase震荡混匀,离心;
S2.2、在装有PCR预混液的8联管中的前5个孔中分别加入2μl步骤S2.1中离心后的混合液;
S2.3、盖紧管盖,震荡混匀,离心,进行PCR扩增;扩增条件为:
1)95℃,3min;
2)95℃,30秒,52℃,30秒,72℃,60秒;35个循环;
3)72℃,10min;
S3、分别对步骤S2中得到的同一患者的正常粘膜组织的DNA与肿瘤组织的DNA的PCR扩增产物在基因分析仪上按照如下过程进行毛细管电泳:
分别步骤S2.3中得到的8联管的前五个孔中的PCR扩增产物各Iμl加入到新的离心管中,然后向所述新的离心管中加入0.5ul LIZ500和10ul HIDI,震荡混匀,离心,在基因分析仪上进行毛细管电泳,得到检测结果。
2.根据权利要求1所述的MSI检测方法,其特征在于,所述扩增引物的序列如下:
针对Bat25的扩增引物为:
BAT-25F:FAM-ATGGAGGATGACGAGTTG;
BAT-25R:CAGGCTTGTACTTTACAGC;
针对Bat26的扩增引物为:
BAT-26F:HEX-ATTGGATATTGCAGCAGTCA;
BAT-26R:GCTCCTTTATAAGCTTCTTCAG;
针对D2S123的扩增引物为:
D2S123F:FAM-AAACAGGATGCCTGCCTTTA;
D2S123R:CTGACTTGGATACCATCTATCT;
针对D17S250的扩增引物为:
D17S250F:HEX-CAGGCTGGTCTCAAACTC;
D17S250R:GGAAGAATCAAATAGACAAT;
针对D5S346的扩增引物为:
D5S346F:TMR-TGGTTTCCATTGTAGCATCTTGAC;
D5S346R:CTGGTTGTTTCCGTAGTATATG。
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