CN116754765A - 一种快速检测棘形红细胞的检测试纸及试剂盒 - Google Patents
一种快速检测棘形红细胞的检测试纸及试剂盒 Download PDFInfo
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Abstract
本发明公开了一种快速检测棘形红细胞的检测试纸及试剂盒,所述检测试纸由支撑板、抗凝样品垫、连接垫、反应显色区和吸收垫组成;其中,所述抗凝样品垫和所述吸收垫分别设置于所述支撑板上且位于支撑板上相对的两侧,所述抗凝样品垫通过连接垫连接至反应显色区的一端,所述反应显色区的另一端与吸收垫相连。本发明主要是基于检测红细胞表面结合的双链DNA来鉴定棘形红细胞的胶体金检测方法,运用本发明的检测试纸及试剂盒进行检测的方法无需特定的场地和设备即可实现,简单快速,准确度高。本发明的检测试纸及试剂盒可用于一切通过检测棘形红细胞进行诊断的临床相关病例。
Description
技术领域
本发明属于生物检测技术领域,具体涉及一种快速检测棘形红细胞的检测试纸及试剂盒。
背景技术
棘形红细胞(acanthocyte)是特指红细胞表面有针尖状突起,其间距不规则,突起的长度和宽度不一,一般在β脂蛋白缺乏症患者的血涂片中出现较多,也可见于脾切除后、酒精中毒性肝脏疾病。南通大学课题组近来研究发现在高原红细胞增多小鼠模型血液中也出现不同程度棘形红细胞增多的现象。
现有的棘形红细胞检测技术主要依赖显微镜对其进行形态进行观察分辨,其检测方法的应用受到设备和场地的限制。
发明内容
针对现有技术的不足,本发明提供一种快速检测棘形红细胞的检测试纸及试剂盒,通过测定红细胞表面的核酸来反映红细胞棘形变化。
本发明是通过以下技术方案实现的:
一种快速检测棘形红细胞的检测试纸,所述检测试纸由支撑板、抗凝样品垫、连接垫、反应显色区和吸收垫组成;其中,所述抗凝样品垫和所述吸收垫分别设置于所述支撑板上且位于支撑板上相对的两侧,所述抗凝样品垫通过连接垫连接至反应显色区的一端,所述反应显色区的另一端与吸收垫相连;
所述抗凝样品垫为含有EDTA-K2抗凝剂的Leukosorb纤维素膜;
所述连接垫为胶体金垫,所述胶体金垫上附有结合了CD47抗体的胶体金颗粒;
所述反应显色区包括硝酸纤维素膜、检测线和质检线;所述硝酸纤维素膜为荧光法NC膜;所述检测线处含有不能穿透活细胞细胞膜的核酸染料;所述质检线处含有CD47的二抗抗体。
优选地,所述硝酸纤维素膜的孔径为8μm。
优选地,所述不能穿透活细胞细胞膜的核酸染料为碘化丙啶。
优选地,所述吸收垫为吸水纸。
一种快速检测棘形红细胞的试剂盒,包括上述的检测试纸,以及一次性末梢采样针、含1mL抗凝稀释液的样品采样管、迷你便携式紫外灯。
一种用于诊断高原红细胞增多症的试剂盒,包括上述的检测试纸,以及一次性末梢采样针、含1mL抗凝稀释液的样品采样管、迷你便携式紫外灯。
本发明的有益效果如下:
本发明主要是基于检测红细胞表面结合的双链DNA来鉴定棘形红细胞的胶体金检测方法,运用本发明的检测试纸及试剂盒进行检测的方法无需特定的场地和设备即可实现,简单快速,准确度高。本发明的检测试纸及试剂盒可用于一切通过检测棘形红细胞进行诊断的临床相关病例。
附图说明
图1为实施例1中红细胞形态变化的扫面电镜图;
图2为实施例2中红细胞mtDNA含量变化的PCR检测图;
图3为实施例3中红细胞表面核酸阳性细胞群变化的流式细胞术检测图;
图4为实施例4中快速检测棘形红细胞的检测试纸的结构示意图;
图4中:1、支撑板;2、抗凝样品垫;3、连接垫;4、反应显色区;5、吸收垫;6、硝酸纤维素膜;7、检测线;8、质检线;
图5为实施例4中快速检测棘形红细胞的试剂盒的操作示意图;
图6为实施例4中快速检测棘形红细胞的试剂盒的检测结果判断示意图。
具体实施方式
下面结合附图与具体实施例对本发明做进一步详细说明。
以下实施例中所用小鼠品种为C57BL/6N,由南通大学动物中心提供。
实施例1高原暴露前后小鼠血液中红细胞棘形变化情况
采用动物低压低氧设备模拟海拔6000米高原暴露小鼠模型。小鼠经过持续暴露7和14天后,获取小鼠血液并制备红细胞样本,采用扫描电镜观察小鼠血液中红细胞的形态。红细胞电镜样品准备及拍摄具体操作方法如下:
小鼠麻醉后采用眼球采血收集血液至EDTA抗凝管中,500g离心5min,分离血浆与红细胞。弃去上层血浆,再用等渗的PBS将下层红细胞清洗三遍。随后将样品用含2.5%(v/v)戊二醛的PBS溶液按体积比1:10比例进行固定过夜。红细胞固定好后,用纯水将固定后的红细胞清洗三遍,再将清洗后的红细胞转移到硅片上,并使用真空干燥机在37℃恒温条件下干燥过夜。最后再使用溅射涂布机将所有样品涂上10nm厚的金膜,通过扫描电镜放大3000倍进行拍摄。
结果如图1所示,与常压常氧(NN)对照组相比,低压低氧暴露7天(HH-7d)和14天(HH-14d)组的小鼠红细胞棘形明显增多,100nM CpGDNA处理组为阳性对照组。
实施例2高原暴露前后小鼠血液中红细胞表面mtDNA含量变化情况
为了验证高原暴露后红细胞是否存在线粒体DNA(mtDNA),在模拟高原6000米持续暴露7和14天后,收集小鼠红细胞后提取红细胞核酸样本,采用PCR对mtDNA相关基因(ND1、COX1、16S)进行检测。红细胞膜核酸样品提取及PCR具体操作方法如下:
小鼠麻醉后采用眼球采血收集血液至EDTA抗凝管中,500g离心5min,分离血浆与红细胞。弃去上层血浆,并用等渗的PBS将下层红细胞清洗三遍。采用红血球计数仪对红细胞进行计数。取约1×108个红细胞裂红处理后,收集沉淀,加入10μL的YSY Buffer(南京尧顺禹生物科技有限公司)裂解沉淀物。裂解程序为65℃处理30min、95℃处理5min、16℃处理1min。吸取裂解后的上清溶液2μL加至八联排管中,再向每管中加入10μL的2×Rapid TaqMaster Mix、1μL各线粒体基因的上游引物、1μL的各线粒体基因下游引物和6μL的RNA free水。待样品充分混悬后放入PCR扩增仪,设置95℃预变性3min、95℃变性15s、60℃退火15s、72℃延伸15s,重复35个循环后,72℃延伸5min。PCR扩增结束后,使用2%的琼脂糖凝胶,取10μL样品进行凝胶电泳并拍照观察。
结果如图2所示,与常压常氧(NN)对照组相比,低压低氧暴露7天(HH-7d)和14天(HH-14d)组的小鼠红细胞的mtDNA含量明显增多,表明高原暴露后红细胞表面mtDNA增多引起红细胞变成棘形增多。
实施例3在体水平检测红细胞表面DNA含量
为了进一步在在体水平上检测红细胞表面上存在DNA,本实施例在模拟高原6000米持续暴露7和14天后,收集小鼠红细胞并用碘化丙啶(PI)对样本进行孵育,采用流式细胞术对红细胞表面核酸阳性细胞进行分析。红细胞表面PI染色及流式细胞术具体操作方法如下:
小鼠麻醉后采用眼球采血收集血液至EDTA抗凝管中,500g离心5min,分离血浆与红细胞。弃去上层血浆,并用等渗的PBS将下层红细胞清洗三遍。取1×107个红细胞,加入100μL的2%小鼠血清封闭20min,按1:20加入100μL的PI染料(50μg/mL,PROTEINBIO,PDA0022),重悬,冰上避光孵育30min。结束后,FL3(488nm/620nm)通道流式上机检测。
结果如图3所示,与常压常氧(NN)对照组相比,低压低氧暴露7天(HH-7d)和14天(HH-14d)组的小鼠红细胞表面核酸阳性占比细胞数明显增多,表明高原暴露后红细胞表面结合的双链DNA含量明显增多。
实施例4
以上实施例1-3的实验结果表明:高原(低压低氧)暴露后红细胞表面DNA(尤其是mtDNA)含量明显增多,可进一步促进红细胞棘形变化。为了进一步简化红细胞棘形检测的方法,本实施例提供一种快速检测棘形红细胞的检测试纸及试剂盒,用于快速检测正常人是否有罹患高原红细胞增多症的风险,其原理为通过测定红细胞表面的核酸来反映红细胞棘形变化。
一种快速检测棘形红细胞的检测试纸,如图4所示,所述检测试纸由支撑板1、抗凝样品垫2、连接垫3、反应显色区4和吸收垫5组成,其中,所述抗凝样品垫2和所述吸收垫5分别设置于所述支撑板1上且位于支撑板1上相对的两侧,所述抗凝样品垫2通过连接垫3连接至反应显色区4的一端,所述反应显色区4的另一端与吸收垫5相连。
所述抗凝样品垫2为含有EDTA-K2抗凝剂的Leukosorb纤维素膜,可去除全血中的白细胞。所述连接垫3为胶体金垫,所述胶体金垫上附有结合了CD47抗体的胶体金颗粒。所述吸收垫5为吸水纸。
如图4所示,所述反应显色区4包括硝酸纤维素膜6、检测线7和质检线8;所述硝酸纤维素膜6为孔径8μm的荧光法NC膜;所述检测线7处含有不能穿透活细胞细胞膜的核酸染料,如碘化丙啶;所述质检线8处含有CD47的二抗抗体。
检测原理:当红细胞表面结合含CpG序列的DNA后,会引起红细胞棘形变化,此时棘形红细胞表面可被核酸染料(检测线7)结合,在紫外照射下显示出核酸阳性;而棘形红细胞表面的CD47由于构象改变,无法被CD47抗体(质检线8)检测出,显示为阴性。同时由于正常红细胞表面不含有CpG序列的DNA,所以核酸染料(检测线7)孵育后无法检测出红细胞表面的核酸,显示为阴性;而正常红细胞表面的CD47构象正常,因此会被CD47抗体(质检线8)检测出,显示为阳性。综上,无论人体内是否存在棘形红细胞,正常红细胞相比于棘形红细胞必定占大多数,因此质检线8必然显色,而只有存在棘形红细胞的情况下检测线7才会显色,正常红细胞不会使检测线7显色。基于此,就能测出血液内是否存在棘形红细胞,进而判断正常人是否有罹患高原红细胞增多症的风险。
一种快速检测棘形红细胞的试剂盒,包括上述的检测试纸(装在铝箔袋中),以及一次性末梢采样针、含1mL抗凝稀释液的样品采样管、迷你便携式紫外灯和说明书。
上述快速检测棘形红细胞的试剂盒的操作方法,如图5所示,具体步骤如下:
(1)血液样品获得:取一次性末梢采样针在无名指处取1滴血液滴至样品采样管内并颠倒混匀。
(2)取出铝箔袋内的检测试纸,在抗凝样品垫处滴入5滴稀释后的样品液,放平静置15min。
(3)打开迷你便携式紫外灯对反应显色区进行照射,对比说明书读取检测结果,如图6所示,其中质检线(C)不显色则为无效,质检线(C)显色时,检测线(T)显色则为阳性,不显色则为阴性。
以上仅为本发明的部分优选实施例,无论是文字还是附图都不能因此限制本发明的保护范围,凡是在与本发明一个整体的构思下,利用本发明说明书及附图内容所作的等效结构变换,或直接/间接运用在其他相关的技术领域均应包括在本发明的保护范围内。
Claims (6)
1.一种快速检测棘形红细胞的检测试纸,其特征在于,所述检测试纸由支撑板、抗凝样品垫、连接垫、反应显色区和吸收垫组成;其中,所述抗凝样品垫和所述吸收垫分别设置于所述支撑板上且位于支撑板上相对的两侧,所述抗凝样品垫通过连接垫连接至反应显色区的一端,所述反应显色区的另一端与吸收垫相连;所述抗凝样品垫为含有EDTA-K2抗凝剂的Leukosorb纤维素膜;
所述连接垫为胶体金垫,所述胶体金垫上附有结合了CD47抗体的胶体金颗粒;所述反应显色区包括硝酸纤维素膜、检测线和质检线;所述硝酸纤维素膜为荧光法NC膜;所述检测线处含有不能穿透活细胞细胞膜的核酸染料;所述质检线处含有CD47的二抗抗体。
2.根据权利要求1所述的一种快速检测棘形红细胞的检测试纸,其特征在于,所述硝酸纤维素膜的孔径为8μm。
3.根据权利要求1所述的一种快速检测棘形红细胞的检测试纸,其特征在于,所述不能穿透活细胞细胞膜的核酸染料为碘化丙啶。
4.根据权利要求1所述的一种快速检测棘形红细胞的检测试纸,其特征在于,所述吸收垫为吸水纸。
5.一种快速检测棘形红细胞的试剂盒,其特征在于,包括如权利要求1-4任一项所述的检测试纸,以及一次性末梢采样针、含1mL抗凝稀释液的样品采样管、迷你便携式紫外灯。
6.一种用于诊断高原红细胞增多症的试剂盒,其特征在于,包括如权利要求1-4任一项所述的检测试纸,以及一次性末梢采样针、含1mL抗凝稀释液的样品采样管、迷你便携式紫外灯。
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