CN113667723B - 一种基于CRISPR/Cas12a与链置换扩增反应的PSA检测方法 - Google Patents
一种基于CRISPR/Cas12a与链置换扩增反应的PSA检测方法 Download PDFInfo
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Abstract
本发明涉及一种基于CRISPR/Cas12a与链置换扩增反应的PSA检测方法,首先通过基于核酸适配体的链置换扩增反应,将PSA的信号转换为链置换扩增反应产物DNA的信号,同时链置换扩增反应产物DNA激活CRISPR/Cas12a系统的反向酶切功能,并将连接两种金纳米颗粒的单链DNA切成含2‑4个碱基的片段,使金纳米颗粒由聚集变为分散状态,溶液的颜色表现为紫色向红色的变化。本发明对于PSA检测,通过肉眼可见的颜色变化,紫外可见吸收光谱和动态光散射(DLS)研究了该方法的可行性。
Description
技术领域
本发明属于生物传感器的开发方法,涉及一种基于CRISPR/Cas12a与链置换扩增反应的PSA检测方法。
背景技术
目前为止,已开发出许多检测PSA的方法,如荧光光谱法(Clin Cancer Res 2019,25(1),177)、基于化学发光的免疫检测法(Luminescence 2017,32(8),1547)、酶联免疫吸附检测法(ELISA)(Biosens Bioelectron 2015,69,128)、电化学发光法(Sens.ActuatorsB Chem.2020,315,128155)、表面增强荧光免疫酶法(ACS Nano 2017,11(5),4926)和表面增强拉曼散射法(Biosens Bioelectron 2018,119,126)。然而,这些方法大多基于免疫反应,存在一些不足,比如需要昂贵且易变性的抗体、专业的技术人员和特定的仪器设备,这为PSA的检测增加了难度。另外,本课题组前期申请的“一种超灵敏检测前列腺特异性抗原PSA的方法”发明专利,目前已在公开阶段,公开号CN112301095A,中国。该发明专利联合聚合酶链扩增技术(PCR)与动态光散射技术,通过监测金纳米颗粒(AuNPs)的直径变化实现PSA的定量与超灵敏检测。该专利通过引入核酸适配体避免了抗体的使用,但由于需要使用PCR技术,仍然需要专业的技术人员的专业操作才能实现。此外,该发明专利与现申请专利的区别表现在:(1)PCR技术是一种变温放大技术,需要依赖特定的仪器设备,而现申请专利中的放大技术是等温放大技术,反应条件温和,室温下即可进行,不需要额外的仪器设备;(2)该发明专利是通过金纳米颗粒的直径变化来定量PSA,而本发明专利是通过金纳米溶液的吸收光谱的变化来定量PSA,并且可通过肉眼观察颜色的变化初步判断PSA含量的高低。新发明避免了昂贵且易变性的抗体的使用,降低了检测过程对专业仪器设备与专业操作人员的依赖性,使PSA的检测不再局限于实验室等特定场合。
发明内容
要解决的技术问题
为了避免现有技术的不足之处,本发明提出一种基于CRISPR/Cas12a与链置换扩增反应的PSA检测方法,通过链置换扩增反应将PSA的检测转化为DNA的检测降低检测成本;CRISPR/Cas12a系统与链置换扩增反应联合应用提高PSA的检测灵敏度;引入与AuNPs实现可视化检测PSA,降低现有检测技术对仪器的依赖性。
技术方案
一种基于CRISPR/Cas12a与链置换扩增反应的PSA检测方法,其特征在于DNA序列表如下:
其中:n是2-10中的随机数字,x是6-20中的随机数字,y是1-50中的随机数字;PSA检测步骤如下:
步骤1、制备修饰了DNA的金纳米粒子:将巯基修饰的DNA:Oligo 6和Oligo 7分别溶解于60.0μL的pH为5.0的醋酸盐缓冲溶液,再加入12.0μL的20.0mM的三(2-羧乙基)膦TCEP,以还原修饰在DNA上的巯基为二硫键;然后将上述溶液全部转移至10.0mL AuNPs溶液中,室温孵育16.0小时;
然后在接下来的44.0小时内,向金纳米粒子溶液中加入5.0M的氯化钠NaCl溶液,使最终溶液中NaCl的浓度为100.0mM;
修饰完成,将溶液离心处理三次,除去未修饰到金纳米粒子表面的DNA,每次离心后用pH 8.0的三羟甲基氨基甲烷-醋酸Tris-Ac缓冲溶液洗涤;
得到修饰了DNA的金纳米粒子;
步骤2、基于核酸适配体的链置换扩增反应:将发卡结构的DNA,Oligo 3在85.0~105.0℃的条件下加热不少于5.0分钟,然后自然冷却至室温;将被测溶液与适配体:Oligo2,在20.0~40.0℃孵育不少于5.0分钟,在20.0~40.0℃条件下继续延伸反应;
被测溶液与适配体即Oligo 2在缓冲溶液中20.0~40.0℃反应不少于5.0分钟,然后加入克列诺片段聚合酶,按1:10:10的比例加入Oligo 1、Oligo 3、Oligo 4和过量的dNTPs,20.0~40.0℃条件下进行不少于30分钟的扩增反应;
扩增反应的产物离心后取上清液;
步骤3、CRISPR/Cas12a反向酶切功能激活的反应:
Cas12a/crRNA反应液中含有但不仅限于NaCl、Tris-HCl、MgCl2和牛胎血清蛋白;向反应液中按5:6的比例加入Cas 12a与crRNA,再加入Oligo 5,20.0~40.0℃孵育不少于5分钟,再加入步骤2得到的上清液,20.0~40.0℃反应不少于30分钟得到反应液;
步骤4、显色反应及吸收光谱的测量:向步骤3的反应液中加入步骤1制备的修饰了DNA的金纳米粒子的溶液,若溶液出现的向红色方向变色情况,证明被测溶液中存在PSA。
当检测到PSA时,采用紫外可见吸收光谱测量溶液的吸收光谱与吸光度。
所述溶液的PSA的浓度与吸光度,在PSA的浓度在0.1~5.0ng/mL范围时,PSA的浓度与吸光度为线性关系,A=0.1285+0.09281C,A为吸光度,C为PSA浓度,单位为C:ng mL-1。,在PSA的浓度在0.1~5.0ng/mL范围时,PSA的浓度与吸光度为线性关系,A=0.1285+0.09281C,A为吸光度,C为PSA浓度,单位为C:ng mL-1。
所述步骤1的离心处理以不少于10000.0rpm转速的离心机处理,每次离心不少于15.0分钟。
所述步骤2的离心速度为不少于1000.0转速。
所述金纳米粒子的制备:将柠檬酸钠溶液快速加入煮沸的氯金酸溶液中搅拌,待溶液颜色由淡黄色变为酒红色后,再回流搅拌,通过连续搅拌将溶液冷却到室温,并储存在4.0℃的冰箱中。
所述本发明中用到的序列由生工生物技术有限公司合成。
有益效果
本发明提出的一种基于CRISPR/Cas12a与链置换扩增反应的PSA检测方法,首先通过基于核酸适配体的链置换扩增反应,将PSA的信号转换为链置换扩增反应产物DNA的信号,同时链置换扩增反应产物DNA激活CRISPR/Cas12a系统的反向酶切功能,并将连接两种金纳米颗粒的单链DNA切成含2-4个碱基的片段,使金纳米颗粒由聚集变为分散状态,溶液的颜色表现为紫色向红色的变化。
本发明对于PSA检测,通过肉眼可见的颜色变化,紫外可见吸收光谱和动态光散射(DLS)研究了该方法的可行性。如图1A所示,加入10.0ng mL-1PSA前后,溶液颜色变化显著,在没有PSA的情况下,溶液中的金纳米颗粒聚集沉降并使溶液颜色呈现淡紫色,加入10.0ngmL-1PSA后,溶液中的金纳米颗粒不聚集,溶液颜色是红色。紫外可见吸收光谱也表明随着PSA的加入,溶液的吸光度呈现明显的增加,表明金纳米颗粒从聚集状态变为分散状态(图1B)。DLS图像进一步证实这一结果,加入10.0ng mL-1PSA前,金纳米颗粒水合直径大,颗粒处于聚集状态,加入10.0ng mL-1PSA后,金纳米颗粒水合直径小,颗粒处于分散状态(图1C)。这些结果表明,该方法可以通过测量AuNPs的吸收光谱的变化来定量PSA。
对分析物的线性响应是分析的关键,因此该方法测定了不同浓度PSA存在时球形核酸溶液吸收光谱的变化情况,并以522nm处吸收光谱值为判断依据。由图2可知,随着PSA浓度的增加,522nm处球形核酸的吸收值逐渐增加,结果显示在0.1ng mL-1到5.0ng mL-1范围内,球形核酸的紫外吸收随PSA浓度的线性减小。线性回归方程A(吸光度)=0.1285+0.09281C(PSA)(C:ng mL-1)和检出限28.0pg mL-1(3δ/斜率)。这些结果表明,该生物传感器能够实现PSA的高灵敏度检测。
附图说明
图1:(A)溶液的颜色;(B)吸收光谱的结果;(C)动态光散射的结果;(a)没有加入PSA的结果;(b)加入10.0ng mL-1PSA的结果。
图2:溶液的吸光度与PSA浓度变化的线性关系,PSA的浓度从0.1ng mL-1到5.0ngmL-1。误差棒表示三次测量值的标准差。
具体实施方式
现结合实施例、附图对本发明作进一步描述:
本方法实施例所需要的DNA序列列于表1-1,由生工生物技术有限公司合成,具体如下表:
1-1本发明中用到的序列
注:n是2-10中的随机数字,x是6-20中的随机数字,y是1-50中的随机数字。
本方法实施例的操作步骤如下:
步骤1:金纳米粒子(AuNPs)的制备(参考已有的方法)。将2.0mL 38.8mM柠檬酸钠溶液快速加入煮沸的1.0mM氯金酸(HAuCl4)溶液中搅拌,待溶液颜色由淡黄色变为酒红色后,再回流搅拌15.0分钟,通过连续搅拌将溶液冷却到室温。最后AuNPs通过0.4μm尼龙滤膜过滤收集并储存在4.0℃的冰箱待用。
接着制备脱氧核糖核酸(DNA)修饰的AuNPs:首先,将巯基修饰的DNA(Oligo6和Oligo 7)分别溶解于60.0μL的pH为5.0的醋酸盐缓冲溶液,再加入12.0μL的20.0mM的三(2-羧乙基)膦(TCEP),以还原修饰在DNA上的巯基为二硫键,然后将上述溶液全部转移至10.0mL AuNPs溶液中,室温孵育16.0小时。
然后在接下来的44.0小时内,向金纳米粒子溶液中加入5.0M的氯化钠(NaCl)溶液,并使最终溶液中NaCl的浓度为100.0mM。修饰完成。
将溶液放置在13800.0rpm转速的离心机离心三次,每次离心30.0分钟,以除去未修饰到金纳米粒子表面的DNA(每次用pH 8.0的三羟甲基氨基甲烷-醋酸(Tris-Ac)缓冲溶液洗涤)。最后将制备的修饰了DNA的金纳米粒子(球形核酸)稀释到所需浓度,储存于4.0℃冰箱于步骤3中使用。
步骤2:基于核酸适配体的链置换扩增反应。当PSA不存在时,Oligo 1和Oligo 2为DNA/DNA双链结构,无法引发链替换扩增反应,反应终止。当PSA存在时,PSA与适配体Oligo2结合释放引物Oligo 1,释放的Oligo 1因与发卡DNA Oligo 3互补而打开发卡形成DNA/DNA双链结构,引物Oligo 4与打开的Oligo 3序列部分互补形成双链,并在克列诺片段聚合酶的作用下以彼此为模板延伸,得到两条更长的DNA链,并释放引物Oligo 1。释放的Oligo1可以继续打开发卡Oligo 3实现链替换扩增反应,而新生成的长DNA链在步骤(3)中可激发CRISPR/Cas12a系统的反向切割活性,影响溶液中AuNPs的颜色和吸收信号的变化,将PSA的检测转化为DNA的检测,通过观察颜色变化与测定吸收信号的变化可以定量PSA。
基于核酸适配体的链置换扩增反应条件:将发卡结构的DNA(Oligo 3)在85.0~105.0℃的条件下加热5.0分钟,然后自然冷却至室温。将被测液体与适配体(Oligo 2)在20.0~40.0℃孵育不少于5.0分钟,在20.0~40.0℃条件下继续延伸反应;
被测溶液与适配体(Oligo 2)在含有20.0mM Tris-OAc(pH 7.9)、50.0mM KOAc、10.0mM Mg(OAc)2和1.0mM DTT的溶液中,20.0~40.0℃反应不少于5.0分钟,,然后加入0.5U克列诺片段聚合酶、2.5μL 0.1μM Oligo 1、2.5μL 1.0μM Oligo 3、2.5μL1.0μM Oligo4和2.5μL 10.0mM dNTPs,,20.0~40.0℃条件下进行不少于30分钟的扩增反应;
扩增反应的产物在桌面离心机以10000.0rpm的速度离心1分钟,取上清液于步骤(3)中使用。
步骤3:CRISPR/Cas12a反向酶切功能的激活。Cas12a蛋白(LbCpf1、FnCpf1、AsCpf1)同时具有正向(cis)和反向(trans)切割单链DNA的活性。当Cas12a与特定的crRNA及其目标DNA形成三元复合物时,该复合物获得强大反向切割活性,并将单链DNA切成2-4个核苷酸片段。利用Cas12a的这种性质,首先通过链替换扩增反应将PSA的信号检测转换为DNA的信号检测,然后链替换扩增的双链DNA产物与设计的crRNA(Oligo 8)互补并激发Cas12a的反向切割活性。
CRISPR/Cas12a反向酶切功能激活的反应条件:20μL pH为7.9的Cas12a/crRNA反应液中含有100.0mM NaCl、50.0mM Tris-HCl、10.0mM MgCl2和100.0μg mL-1牛胎血清蛋白(BSA)。反应液中加入100.0nM Cas 12a、120.0nM crRNA和400.0nM Oligo 5,20.0~40.0℃孵育不少于5分钟;再加入5.0μL步骤(2)中的上清液,20.0~40.0℃反应不少于30分钟得到反应液;留作步骤4中使用。
步骤4:显色反应及吸收光谱的测量。因Oligo 5可分别与Oligo 6和Oligo 7互补形成双螺旋而拉近Oligo 6和Oligo 7修饰的金纳米颗粒的距离,使金纳米颗粒由分散状态变为聚集状态,溶液颜色呈现紫色,但是激活的Cas12a可以将Oligo 5切成2-4个碱基的片段,使金纳米颗粒不聚集,溶液颜色保持红色。
显色反应及吸收光谱的测量条件:分别向步骤(3)的反应液中加入25.0μL 0.34nM(1)中制备的球形核酸,等待10分钟,通过肉眼观察溶液的变色情况,再用紫外可见吸收光谱测量溶液的吸收光谱。
在PSA的浓度在0.1~5.0ng/mL范围时,PSA的浓度和吸光度满足线性变化,采用线性回归方程A(吸光度)=0.1285+0.09281C(PSA浓度)能够得到在0.1~5.0ng/mL范围内的两者关系,浓度单位C:ng mL-1)。这些结果表明,该生物传感器能够实现PSA的高灵敏度检测。
实例一:通过加标回收率研究该检测方法在复杂生物体质中的检测能力,即在没有被测物质的样品基质中加入定量的标准物质,按样品的处理步骤分析,得到的结果与理论值的比值。具体过程如下:首先,向链置换扩增反应的缓冲液中加入5%人血清和0.2ngmL-1的标准PSA样品,经过链置换扩增反应,用本发明方法检测到的PSA浓度是0.22ng mL-1,回收率是110.0%,相对标准偏差(RSD)是3.6%。
实例二:通过加标回收率研究该检测方法在复杂生物体质中的检测能力,即在没有被测物质的样品基质中加入定量的标准物质,按样品的处理步骤分析,得到的结果与理论值的比值。具体过程如下:首先,向链置换扩增反应的缓冲液中加入5%人血清和1.0ngmL-1的标准PSA样品,经过链置换扩增反应,用本发明方法检测到的PSA浓度是1.12ng mL-1,回收率是111.9%,相对标准偏差(RSD)是1.8%。
实例三:通过加标回收率研究该检测方法在复杂生物体质中的检测能力,即在没有被测物质的样品基质中加入定量的标准物质,按样品的处理步骤分析,得到的结果与理论值的比值。具体过程如下:首先,向链置换扩增反应的缓冲液中加入5%人血清和5.0ngmL-1的标准PSA样品,经过链置换扩增反应,用本发明方法检测到的PSA浓度是5.01ng mL-1,回收率是100.2%,相对标准偏差(RSD)是2.2%。
Claims (6)
1.一种基于CRISPR/Cas12a与链置换扩增反应的前列腺特异性抗原PSA检测方法,其特征在于DNA序列表如下:
其中:n是2-10中的随机数字,x是6-20中的随机数字,y是1-50中的随机数字;
前列腺特异性抗原PSA检测步骤如下:
步骤1、制备修饰了DNA的金纳米粒子:将巯基修饰的DNA:Oligo 6和Oligo 7分别溶解于60.0μL的pH为5.0的醋酸盐缓冲溶液,再加入12.0μL的20.0mM的三(2-羧乙基)膦TCEP,以还原修饰在DNA上的巯基为二硫键;然后将上述溶液全部转移至10.0mL AuNPs溶液中,室温孵育16.0小时;
然后在接下来的44.0小时内,向金纳米粒子溶液中加入5.0M的氯化钠NaCl溶液,使最终溶液中NaCl的浓度为100.0mM;
修饰完成,将溶液离心处理三次,除去未修饰到金纳米粒子表面的DNA,每次离心后用pH 8.0的三羟甲基氨基甲烷-醋酸Tris-Ac缓冲溶液洗涤;
得到修饰了DNA的金纳米粒子;
步骤2、基于核酸适配体的链置换扩增反应:将发卡结构的DNA,Oligo 3在85.0-105.0℃的条件下加热不少于5.0分钟,然后自然冷却至室温;将被测溶液与适配体:Oligo 2,在20.0-40.0℃孵育不少于5.0分钟,在20.0-40.0℃条件下继续延伸反应;
被测溶液与适配体即Oligo 2在缓冲溶液中20.0-40.0℃反应不少于5.0分钟,然后加入克列诺片段聚合酶,按1:10:10的比例加入Oligo 1、Oligo 3、Oligo 4和过量的dNTPs,20.0-40.0℃条件下进行不少于30分钟的扩增反应;
扩增反应的产物离心后取上清液;
步骤3、CRISPR/Cas12a反向酶切功能激活的反应:
Cas12a/crRNA反应液中含有但不仅限于NaCl、Tris-HCl、MgCl2和牛胎血清蛋白;向反应液中按5:6的比例加入Cas 12a与crRNA,再加入Oligo 5,20.0-40.0℃孵育不少于5分钟,再加入步骤2得到的上清液,20.0-40.0℃反应不少于30分钟得到反应液;
步骤4、显色反应及吸收光谱的测量:向步骤3的反应液中加入步骤1制备的修饰了DNA的金纳米粒子的溶液,若溶液出现的向红色方向变色情况,证明被测溶液中存在前列腺特异性抗原PSA。
2.根据权利要求1所述基于CRISPR/Cas12a与链置换扩增反应的前列腺特异性抗原PSA检测方法,其特征在于:当检测到PSA时,采用紫外可见吸收光谱测量溶液的吸收光谱与吸光度。
3.根据权利要求2所述基于CRISPR/Cas12a与链置换扩增反应的前列腺特异性抗原PSA检测方法,其特征在于:所述溶液的PSA的浓度与吸光度,在PSA的浓度在0.1~5.0ng/mL范围时,PSA的浓度与吸光度为线性关系,A=0.1285+0.09281C,A为吸光度,C为PSA浓度,单位为C:ng mL-1。
4.根据权利要求1所述基于CRISPR/Cas12a与链置换扩增反应的前列腺特异性抗原PSA检测方法,其特征在于:所述步骤1的离心处理以不少于10000.0rpm转速的离心机处理,每次离心不少于15.0分钟。
5.根据权利要求1所述基于CRISPR/Cas12a与链置换扩增反应的前列腺特异性抗原PSA检测方法,其特征在于:所述步骤2的离心速度为不少于1000.0转速。
6.根据权利要求1所述基于CRISPR/Cas12a与链置换扩增反应的前列腺特异性抗原PSA检测方法,其特征在于:所述金纳米粒子的制备:将柠檬酸钠溶液快速加入煮沸的氯金酸溶液中搅拌,待溶液颜色由淡黄色变为酒红色后,再回流搅拌,通过连续搅拌将溶液冷却到室温,并储存在4.0℃的冰箱中。
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