CN113832141B - 粪便人源基因组dna特异性纯化方法及试剂盒 - Google Patents
粪便人源基因组dna特异性纯化方法及试剂盒 Download PDFInfo
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Abstract
本发明属于生物医学领域,具体涉及粪便人源基因组DNA特异性纯化方法及试剂盒,所述试剂盒包括以下组分:ST Buffer、STC buffer、SYS buffer、蛋白酶K溶液、WS1Buffer、WS2Buffer、STE Buffer和纯化柱。本发明所述试剂盒及纯化方法可以减少肠道微生物DNA的干扰和粪便杂质的干扰,纯化的DNA可用于肠癌粪便DNA检测。
Description
技术领域
本发明属于生物医学领域,具体涉及粪便人源基因组DNA特异性纯化方法及试剂盒。
背景技术
WHO公布的2020年最新全球结直肠癌发病和死亡数据显示,结直肠癌是全球发病率排第三位,死亡率排第二位的恶性肿瘤。而在中国,结直肠癌发病率高居恶性肿瘤第二位,死亡率高居恶性肿瘤第三位。且随着中国人群的生活水平的逐渐提高,生活方式逐渐西化,中国结直肠癌的发病率和死亡率呈现逐年升高的趋势。
预防结直肠癌有效方法之一就是进行早期筛查。目前结直肠癌的早期筛查方法有以下几种:1)肠镜,2)粪便隐血,3)粪便DNA检测。肠镜作为肠癌诊断金标准,具有灵敏度高、特异性强等优势,但是其作为一种侵入性的检测手段,痛苦大,患者接受度低,且通量低,难以作为一种初筛手段。粪便隐血操作方便,成本低;但是其灵敏度低,且易受到肠炎、痔疮等干扰;因此在肠癌早筛方面也有一定的局限性。粪便DNA检测是近些年新发展出的肠癌早筛手段,其灵敏度高特异性强的特点使得其正逐渐成为肠癌初筛的主要手段。粪便DNA检测第一步是将粪便中的人源基因组DNA分离纯化,然而粪便中包含着大量的肠道微生物和各种干扰物。如果将粪便中总DNA进行提取,那人源基因组DNA占总DNA的量仅为1/10000~1/1000000,在这样高背景的DNA下去检测人源基因组DNA会导致灵敏低且容易被干扰。同时粪便中干扰物也是粪便人源DNA提取的一个重要难点。
发明内容
基于以上现状和存在的问题,本发明提出了一种粪便人源基因组DNA特异性纯化方法及试剂盒,可用于肠癌粪便DNA检测前的样本预处理。
本发明的目的是开发出一种特异性提取粪便人源基因组DNA的方法,减少肠道微生物DNA的干扰和粪便杂质的干扰,纯化的DNA可用于肠癌粪便DNA检测。
本发明提供的技术方案如下:
一种粪便人源基因组DNA特异性试剂盒,包括以下组分:ST Buffer、STC buffer、SYS buffer、蛋白酶K溶液、WS1 Buffer、WS2 Buffer和STE Buffer。
进一步的,包括以下组分:10~40mL ST Buffer、0.5~3mL STC buffer、0.5~4mLSYS buffer、10~100ul蛋白酶K溶液、0.5~1mL WS1 Buffer、0.5~1mL WS2 Buffer和20~500ul STE Buffer。
进一步的,所述ST Buffer包含0.05~0.5M乙二胺四乙酸二钠,0.01~0.2M Tris-HCl,0.005~0.05M NaCl,0.005~0.05M KCl。
进一步的,所述STC buffer包含0.001~0.05M乙二胺四乙酸二钠,0.001~0.2MTris-HCl,0.001~0.05M NaCl,0.001~0.05M KCl,0.1~10%氯化锂,0.1~10%聚乙烯吡咯烷酮,0.1~10%十二烷基硫酸钠,0.1~10%十六烷基三甲基溴化铵,0.1~2%乙酸钠。
进一步的,所述SYS buffer包含0.001~0.05M乙二胺四乙酸二钠,0.001~0.2MTris-HCl,0.001~0.05M NaCl,4~8M胍盐,胍盐包含盐酸胍或硫氰酸胍中的至少一种,1~20%曲拉通100。
进一步的,所述WS1 Buffer包含0.001~0.05M乙二胺四乙酸二钠,0.001~0.2MTris-HCl,0.001~0.05M NaCl,1~5M胍盐,胍盐包含盐酸胍或硫氰酸胍中的至少一种,1~10%曲拉通100,30~70%醇,所述醇为乙醇或异丙醇中任意一种。
进一步的,所述WS2 Buffer包含0.001~0.05M乙二胺四乙酸二钠,0.001~0.2MTris-HCl,60~90%醇,所述醇为乙醇或异丙醇中任意一种。
进一步的,其特征在于,所述STE Buffer包含0.001~0.05M乙二胺四乙酸二钠,0.001~0.2M Tris-HCl。
一种粪便人源基因组DNA特异性纯化方法,所述方法采用上述试剂盒;
所述方法包括以下步骤:
取ST Buffer至离心管中,再取粪便样品至离心管中,充分震荡混匀;将离心冷冻后,室温融化并震荡混匀,离心后取上清加入STC buffer,再次离心后加入SYS buffer和蛋白酶K,混匀后孵育;
孵育后加入醇,震荡混匀后将混合液加入纯化柱中,离心后倒去收集管中液体;
加入WS1 Buffer于纯化柱中,离心后倒去收集管中液体;
加入WS2 Buffer于纯化柱中,离心后倒去收集管中液体;
更换新的收集管,将纯化柱放置于新的收集管中;
加入STE Buffer于纯化柱中,离心后得到粪便人源基因组DNA。
进一步的,所述的粪便为人源粪便。
一种粪便人源基因组DNA特异性纯化方法,包含以下步骤:
1)取10~40mL ST Buffer至离心管中,所述的ST Buffer包含0.05~0.5M乙二胺四乙酸二钠,0.01~0.2M Tris-HCl,0.005~0.05M NaCl,0.005~0.05M KCl,再取1~10g粪便至离心管中,充分震荡混匀;
2)将离心管放置于-15~-80℃冰箱冷冻不少于2小时;
3)取出冷冻后离心管,室温融化后震荡混匀,5000~10000g离心10-20分钟;
4)取0.1~0.8mL上清至离心管中,加入0.5~3mL STC buffer,所述的STC buffer包含0.001~0.05M乙二胺四乙酸二钠,0.001~0.2M Tris-HCl,0.001~0.05M NaCl,0.001~0.05M KCl,0.1~10%氯化锂,0.1~10%聚乙烯吡咯烷酮,0.1~10%十二烷基硫酸钠,0.1~10%十六烷基三甲基溴化铵,0.1~2%乙酸钠,震荡混匀后10000~20000g离心3~10分钟;
5)转移上清至新离心管中,加入0.5~4mL SYS buffer和10~100ul蛋白酶K,混匀后56~75℃孵育5~30分钟;所述的SYS buffer包含0.001~0.05M乙二胺四乙酸二钠,0.001~0.2M Tris-HCl,0.001~0.05M NaCl,4~8M胍盐,胍盐包含盐酸胍或硫氰酸胍中的至少一种,1~20%曲拉通100,
6)加入0.5~3mL醇,所述醇为乙醇或异丙醇中任意一种,震荡混匀后将混合液加入纯化柱中,5000~20000g离心1~2分钟,倒去收集管中液体;所述纯化柱为核酸离心纯化吸附柱;
7)重复上述步骤至溶液全部转移完毕;
8)加入0.5~1mL WS1 Buffer于纯化柱中,所述的WS1 Buffer包含0.001~0.05M乙二胺四乙酸二钠,0.001~0.2M Tris-HCl,0.001~0.05M NaCl,1~5M胍盐,胍盐包含盐酸胍或硫氰酸胍中的至少一种,1~10%曲拉通100,30~70%醇,所述醇为乙醇或异丙醇中任意一种,5000~20000g离心1~2分钟,倒去收集管中液体;
9)加入0.5~1mL WS2 Buffer于纯化柱中,所述的WS2 Buffer包含0.001~0.05M乙二胺四乙酸二钠,0.001~0.2M Tris-HCl,60~90%醇,所述醇为乙醇或异丙醇中任意一种,5000~20000g离心1~2分钟,倒去收集管中液体;
10)更换新的收集管,将纯化柱放置于新的收集管中;
11)加入20~500ul STE Buffer于纯化柱中,所述STE Buffer包含0.001~0.05M乙二胺四乙酸二钠,0.001~0.2M Tris-HCl,5000~20000g离心1~2分钟,得到粪便人源基因组DNA。
进一步的,ST Buffer采集后的粪便保存温度为不超过37℃,室温保存时间不超过7天。
有益效果
本发明提出了一种粪便人源基因组DNA特异性纯化方法及试剂盒,可用于肠癌粪便DNA检测前的样本预处理。加入乙二胺四乙酸二钠和氯化锂具有提高核酸稳定性,提高核酸回收效率的效果;加入NaCl和十二烷基硫酸钠可以与蛋白等杂质结合,具有去除蛋白杂质的效果;十六烷基三甲基溴化铵具有与多糖等杂质结合的效果,乙酸钠可以调节反应pH值并促进核酸沉淀的作用,因此采用本发明的组分和比例制备的试剂盒提取的DNA浓度相对是最高的,具有最好的技术效果。本发明所述方法可减少肠道微生物DNA的干扰和粪便杂质的干扰,纯化的DNA可用于肠癌粪便DNA检测。
附图说明
图1为该发明试剂盒提取10例粪便保存室温保存7天效果;
图2为20个粪便样本人HBB基因荧光定量PCR扩增曲线;
图3为本发明方法及凯杰QIAamp Fast DNA Stool Mini Kit提取等量粪便样本琼脂糖凝胶图;V1和V2代表本发明方法提取两个样本,Q1和Q2代表凯杰试剂盒提取两个样本;
图4为本发明方法及凯杰QIAamp Fast DNA Stool Mini Kit提取等量粪便样本检测人RPPH1的基因荧光定量PCR扩增曲线1;V代表本发明,Q代表凯杰;
图5为本发明方法及凯杰QIAamp Fast DNA Stool Mini Kit提取等量粪便样本检测人RPPH1的基因荧光定量PCR扩增曲线2。V代表本发明,Q代表凯杰。
具体实施方式
实施例1
取10例粪便样本,分别在室温放置0,1,3,7天后,同时取10例对照粪便,将STBuffer更换成水室温保存2小时候,按照本发明方法和试剂盒进行提取,提取完毕后检测其上述粪便DNA中人源看家基因ACTB。
1)取10mL ST Buffer至离心管中,所述的ST Buffer包含0.1M乙二胺四乙酸二钠,0.01mM Tris-HCl,0.005mM NaCl,0.005mM KCl,再取1g粪便至离心管中,充分震荡混匀;
2)将离心管放置于-15℃冰箱冷冻2小时;
3)取出冷冻后离心管,室温融化后震荡混匀,10000g离心10分钟;
4)取0.1mL上清至离心管中,加入0.5mL STC buffer,所述的STC buffer包含0.001M乙二胺四乙酸二钠,0.001M Tris-HCl,0.001M NaCl,0.001M KCl,0.1%氯化锂,2%聚乙烯吡咯烷酮,2%十二烷基硫酸钠,1%十六烷基三甲基溴化铵,0.1%乙酸钠,震荡混匀后10000g离心3分钟;
5)转移上清至新离心管中,加入0.5mL SYS buffer和20ul蛋白酶K,混匀后60℃孵育5分钟;所述的SYS buffer包含0.001M乙二胺四乙酸二钠,0.001M Tris-HCl,0.001MNaCl,4M盐酸胍,5%曲拉通100,
6)加入0.5mL乙醇,震荡混匀后将混合液加入纯化柱中,10000g离心1分钟,倒去收集管中液体;
7)重复上述步骤至溶液全部转移完毕;
8)加入0.5mL WS1 Buffer于纯化柱中,所述的WS1 Buffer包含0.001M乙二胺四乙酸二钠,0.001M Tris-HCl,0.001M NaCl,1M盐酸胍,1%的曲拉通100,50%乙醇,10000g离心1分钟,倒去收集管中液体;
9)加入0.5mL WS2 Buffer于纯化柱中,所述的WS2 Buffer包含0.001乙二胺四乙酸二钠,0.001M Tris-HCl,75%乙醇,10000g离心1分钟,倒去收集管中液体;
10)更换新的收集管,将纯化柱放置于新的收集管中;
11)加入20ul STE Buffer于纯化柱中,所述STE Buffer包含0.001M乙二胺四乙酸二钠,0.001M Tris-HCl,10000g离心1分钟,得到粪便人源基因组DNA。
结果如图1所示,本发明的试剂盒提取的粪便DNA可以有效检测到人源基因组DNA,且粪便在ST Buffer的保护下室温7天Ct值无明显差异,但是去除ST Buffer后就无法检测到ACTB信号(图1中ct=50代表无荧光信号)。
实施例2
取20例粪便样样本,按照本发明方法和试剂盒进行提取,提取完毕后检测其上述粪便DNA中人源看家基因HBB。
1)取35mL ST Buffer至离心管中,所述的ST Buffer包含0.2M乙二胺四乙酸二钠,0.2mM Tris-HCl,0.01mM NaCl,0.01mM KCl,再取10g粪便至离心管中,充分震荡混匀;
2)将离心管放置于-80℃冰箱冷冻2小时;
3)取出冷冻后离心管,室温融化后震荡混匀,10000g离心20分钟;
4)取0.8mL上清至离心管中,加入2.5mL STC buffer,所述的STC buffer包含0.001M乙二胺四乙酸二钠,0.001M Tris-HCl,0.001M NaCl,0.001M KCl,1%氯化锂,1%聚乙烯吡咯烷酮,10%十二烷基硫酸钠,5%十六烷基三甲基溴化铵,1%乙酸钠,震荡混匀后10000g离心5分钟;
5)转移上清至新离心管中,加入3.5mL SYS buffer和100ul蛋白酶K,混匀后70℃孵育5分钟;所述的SYS buffer包含0.01M乙二胺四乙酸二钠,0.001M Tris-HCl,0.001MNaCl,6M硫氰酸胍,10%曲拉通100,
6)加入2.5mL乙醇,震荡混匀后将混合液加入纯化柱中,10000g离心1分钟,倒去收集管中液体;
7)重复上述步骤至溶液全部转移完毕;
8)加入1mL WS1 Buffer于纯化柱中,所述的WS1 Buffer包含0.001M乙二胺四乙酸二钠,0.001M Tris-HCl,0.001M NaCl,2M硫氰酸胍,2%曲拉通100,50%乙醇,10000g离心1分钟,倒去收集管中液体;
9)加入1mL WS2 Buffer于纯化柱中,所述的WS2 Buffer包含0.001乙二胺四乙酸二钠,0.001M Tris-HCl,80%乙醇,10000g离心1分钟,倒去收集管中液体;
10)更换新的收集管,将纯化柱放置于新的收集管中;
11)加入200ul STE Buffer于纯化柱中,所述STE Buffer包含0.001M乙二胺四乙酸二钠,0.001M Tris-HCl,10000g离心2分钟,得到粪便人源基因组DNA。
20个粪便样本人HBB基因荧光定量PCR扩增曲线结果如图2所示,表明本发明方法可有效的提取粪便中人源基因组DNA,且稳定有效。
实施例3
收集58例结直肠癌粪便样本,13例进展期腺瘤粪便样本,20例小息肉粪便样本,38例正常人粪便样本,按照本发明试剂盒进行人源基因组DNA提取之后,使用苏州唯善生物科技有限公司的转化试剂盒转化纯化,再同时检测粪便中甲基化基因SDC2和SFPR2。
提取步骤如下:
1)取25mL ST Buffer至离心管中,所述的ST Buffer包含0.15M乙二胺四乙酸二钠,0.1mM Tris-HCl,0.01mM NaCl,0.01mM KCl,再取5g粪便至离心管中,充分震荡混匀;
2)将离心管放置于-80℃冰箱冷冻保存至不超过6个月;
3)取出冷冻后离心管,室温融化后震荡混匀,10000g离心10分钟;
4)取0.2mL上清至离心管中,加入0.5mL STC buffer,所述的STC buffer包含0.01M乙二胺四乙酸二钠,0.01M Tris-HCl,0.01M NaCl,0.01M KCl,2%氯化锂,2%聚乙烯吡咯烷酮,2%十二烷基硫酸钠,1%十六烷基三甲基溴化铵,0.5%乙酸钠,震荡混匀后10000g离心3分钟;
5)转移上清至新离心管中,加入0.6mL SYS buffer和20ul蛋白酶K,混匀后70℃孵育10分钟;所述的SYS buffer包含0.01M乙二胺四乙酸二钠,0.01M Tris-HCl,0.01M NaCl,5M盐酸胍,10%曲拉通100,
6)加入0.6mL乙醇,震荡混匀后将混合液加入纯化柱中,10000g离心1分钟,倒去收集管中液体;
7)重复上述步骤至溶液全部转移完毕;
8)加入0.5mL WS1 Buffer于纯化柱中,所述的WS1 Buffer包含0.001M乙二胺四乙酸二钠,0.001M Tris-HCl,0.001M NaCl,2.5M盐酸胍,2.5%的曲拉通100,50%乙醇,10000g离心1分钟,倒去收集管中液体;
9)加入0.5mL WS2 Buffer于纯化柱中,所述的WS2 Buffer包含0.001乙二胺四乙酸二钠,0.001M Tris-HCl,90%乙醇,10000g离心1分钟,倒去收集管中液体;
10)更换新的收集管,将纯化柱放置于新的收集管中;
11)加入100ul STE Buffer于纯化柱中,所述STE Buffer包含0.001M乙二胺四乙酸二钠,0.001M Tris-HCl,10000g离心2分钟,得到粪便人源基因组DNA。
表1.本发明提取的粪便人源基因组DNA甲基化转化后检测SFRP2和SDC2基因灵敏度和特异性。
上述数据说明了本发明的方法可以长期稳定的保护粪便中人源基因组DNA,且通过发明提取的人源基因组DNA可以用于肠癌的无创早期诊断、筛查。
实施例4
采集2份粪便样本,分别取等量粪便使用本发明的方法,具体方法与实施例3一致,及凯杰QIAamp Fast DNA Stool Mini Kit进行核酸提取。提取完毕后分别进行琼脂糖凝胶电泳,并使用荧光定量PCR检测人RPPH1的基因。
由图3可以看出,凯杰试剂盒提取的粪便有一条明显的微生物基因组DNA条带(>5kb),而本发明的试剂盒仅有微量的游离微生物基因组DNA。由图4和5可以看出,本发明的试剂盒可以提取的人基因组DNA扩增后Ct值明显提前于凯杰试剂盒,证明本发明试剂盒提取人源基因组DNA载量远高于凯杰试剂盒。综合图3、4、5可以得出结论,虽然本发明试剂盒在琼脂糖凝胶电泳中无明显DNA条带,这是因为本发明的试剂盒是特异性纯化人源基因组DNA,而人源基因组DNA在粪便中载量仅为粪便微生物的1/10000~1/1000000,因此琼脂糖凝胶电泳无法有效显示,而荧光定量PCR结果充分显示了本发明试剂盒特异性的提取出了粪便人源基因组DNA,且对DNA更稳定的保存,结果更优。
实施例5
分别采集3例粪便,每个粪便等分为5组,组1是如实施例3全配方,组2是去除STCbuffer中的乙二胺四乙酸二钠和氯化锂、组3是去除STC buffer中的NaCl和十二烷基硫酸钠,组4是去除STC buffer中的十六烷基三甲基溴化铵,组5是去除STC buffer中的乙酸钠。分别用上述组分组合本发明方法进行粪便DNA提取,提取完毕后使用Qubit测量各组粪便中DNA含量如表2。
表2.本发明全配方和去除部分组分提取效果测试
样本1(ng/ul) | 样本2(ng/ul) | 样本3(ng/ul) | |
组1 | 14.5 | 25.3 | 10.3 |
组2 | 9.05 | 22.6 | 4.4 |
组3 | 7.74 | 17.7 | 4.34 |
组4 | 3.84 | 16 | 8.19 |
组5 | 11 | 7.3 | 11.5 |
由表2可以看出,加入乙二胺四乙酸二钠和氯化锂具有提高核酸稳定性和提高核酸回收效率的效果;加入NaCl和十二烷基硫酸钠可以与蛋白等杂质结合,具有去除蛋白杂质的效果;十六烷基三甲基溴化铵具有与多糖等杂质结合的效果,乙酸钠可以调节反应pH值并促进核酸沉淀的作用。。在全配方的情况下提取的DNA浓度相对是最高的,证明当前的组分组合是最优组合,去除部分成分会影响到DNA提取的效果。
Claims (1)
1. 一种粪便人源基因组DNA特异性纯化方法,其特征在于,所述方法采用的试剂盒包括以下组分:ST Buffer、STC buffer、SYS buffer、蛋白酶K溶液、WS1 Buffer、WS2 Buffer和STE Buffer;
所述方法包括以下步骤:
1)取10mL ST Buffer至离心管中,所述ST Buffer包含0.1M乙二胺四乙酸二钠,0.01mMTris-HCl,0.005mM NaCl,0.005mM KCl,再取1g粪便至离心管中,充分震荡混匀;
2)将离心管放置于-15℃冰箱冷冻2小时;
3)取出冷冻后离心管,室温融化后震荡混匀,10000g离心10分钟;
4)取0.1mL上清至离心管中,加入0.5mL STC buffer,所述的STC buffer包含0.001M乙二胺四乙酸二钠,0.001M Tris-HCl,0.001M NaCl,0.001M KCl,0.1% 氯化锂,2%聚乙烯吡咯烷酮,2%十二烷基硫酸钠,1%十六烷基三甲基溴化铵,0.1%乙酸钠,震荡混匀后10000g离心3分钟;
5)转移上清至新离心管中,加入0.5mL SYS buffer和20ul 蛋白酶K,混匀后60℃孵育5分钟;所述的SYS buffer包含0.001M乙二胺四乙酸二钠,0.001M Tris-HCl,0.001M NaCl,4M 盐酸胍,5%曲拉通100;
6)加入0.5mL乙醇,震荡混匀后将混合液加入纯化柱中,10000g离心1分钟,倒去收集管中液体;
7)重复上述步骤至溶液全部转移完毕;
8)加入0.5mL WS1 Buffer于纯化柱中,所述的WS1 Buffer 包含0.001M乙二胺四乙酸二钠,0.001M Tris-HCl,0.001M NaCl,1M盐酸胍,1%的曲拉通100,50%乙醇,10000g离心1分钟,倒去收集管中液体;
9)加入0.5mL WS2 Buffer于纯化柱中,所述的WS2 Buffer 包含0.001乙二胺四乙酸二钠,0.001M Tris-HCl,75%乙醇,10000g离心1分钟,倒去收集管中液体;
10)更换新的收集管,将纯化柱放置于新的收集管中;
11)加入20ul STE Buffer于纯化柱中,所述STE Buffer包含0.001M乙二胺四乙酸二钠,0.001M Tris-HCl,10000g离心1分钟,得到粪便人源基因组DNA。
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