CN113009133A - 一种犬细小病毒的检测方法 - Google Patents
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Abstract
本发明提供了一种犬细小病毒的检测方法,该方法包括如下步骤:以单克隆抗体4C11作为包被抗体,铕Eu3+标记的单克隆抗体7D5作为检测抗体,建立了犬细小病毒的双抗体夹心时间分辨荧光免疫分析检测方法。本发明提供的检测方法具有灵敏度高,稳定性好,特异性强,重复性好,操作便捷,受本底因素影响少等优点,适用于CPV的快速检测。
Description
技术领域
本发明涉及病毒检测技术领域,具体的说涉及一种犬细小病毒的检测方法。
背景技术
犬细小病毒感染是由犬细小病毒(Canine parvovirus,CPV)感染引起的,以严重肠炎综合征和心肌炎综合征为特征的犬科和鼬科动物的重要传染病。该病在世界范围内流行,CPV也是我国出入境口岸宠物检疫的重要疫病之一。
传统的CPV检测方法包括血凝和血凝抑制试验、病毒分离鉴定、酶联免疫吸附试验、电镜和免疫电镜观察等。这些方法存在特异性差、灵敏度低、耗时长、操作繁琐等不足之处。胶体金方法尽管可以实现快速检测,但在敏感性和准确性上均有待提高,如需确诊往往需要通过更灵敏和准确的分子生物学方法。分子病毒学方法PCR、套式PCR、荧光定量PCR、LAMP(环介导等温扩增)等方法需要配备价格昂贵的仪器设备,不便于实现现场检疫。
因此,开发一种新的检测方法,快速、敏感、特异、准确地检测犬细小病毒对该疾病的预防和治疗具有重要的意义。
发明内容
本发明提供了一种犬细小病毒的检测方法,该方法包括如下步骤:
以单克隆抗体4C11作为包被抗体,铕Eu3+标记的单克隆抗体7D5作为检测抗体,建立了犬细小病毒的双抗体夹心时间分辨荧光免疫分析检测方法。
具体的说,本发明的一种犬细小病毒的检测方法,包括如下步骤:
(1)固相包被抗体的制备单克隆抗体4C11于微孔板内用2μg/mL包被液(碳酸盐缓冲液,50mmol/L,pH 9.6)包被,每孔100μL,4℃过夜,弃包被液,加封闭液(5%BSA的PBS溶液,pH 7.4),每孔250μL,37℃封闭2h,弃封闭液,用洗涤液(0.5%Tween-20的PBS溶液,pH7.4)洗涤3次,拍干,放于-20℃保存备用;
(2)铕标记检测抗体及其纯化参考铕标记Eu3+试剂盒说明书对检测抗体7D5进行标记。取0.5mg检测抗体7D5,加入到带有滤膜的离心管中,8000×g离心8min;用标记缓冲液(50mmol/L,Na2CO3,pH 9.0)重复洗涤6次;
设置Eu3+标记试剂:标记抗体比例分别为70μL:30μL,60μL:40μL,50μL:50μL,40μL:60μL,30μL:70μL,25℃振荡过夜。标记完成后用Sephadex-G50层析分离纯化,用洗脱液(含50mmol/L Tris-HCL的生理盐水)洗脱,收集流出液(1mL/管),测定每管洗脱液的OD280值,并根据Eu3+标记试剂盒说明书所提供的公式计算标记率;
(3)标准品的制备及标准曲线的绘制用含0.02%BSA,0.05%Proclin300,50mmol/L的Tris-HCL缓冲液(pH 7.8),将CPV抗原配制成浓度为0,5ng/mL,10ng/mL,20ng/mL,40ng/mL,80ng/mL,160ng/mL,640ng/mL的系列标准品溶液,采用BCA法测定CPV抗原浓度,每瓶1mL分装冻干,放-20℃保存备用;使用时分别加入1mL去离子水溶解使用;(4)TRFIA法检测犬细小病毒步骤取25μL标准品或待测样品,加入到包被好的微孔板中,再加入200μL分析缓冲液(8mmol/L NaCL,0.1%明胶,0.05%Proclin300,0.1%mL/L Tween-80的Tris-HCL溶液,pH7.8),25℃振荡孵育1h,弃掉上清液,用洗涤液(0.5%Tween-20的PBS溶液,pH 7.4)洗涤3次,加入步骤(2)中制备得到的150μL Eu3+标记的CPV抗体溶液,再加入100μL分析缓冲液(含0.2%BSA,0.9%NaCL,19.6mg/L DTPA,0.05%Proclin300,0.1%mL/L Tween-20的Tris-HCL 50mmol/L溶液,pH7.8),混均25℃孵育1h,去除上清,洗涤液洗涤5次,加入200μL增强液(0.1%Triton X-100,3.99mg/L β-NTA,19.33mg/LTOPO,1.3g/L邻苯二甲酸氢钾,0.6%冰醋酸),微孔板于干式荧光免疫分析仪上读取荧光值。
本发明提供的犬细小病毒的检测方法(简称TRFIA法)是利用一株犬细小病毒的单克隆抗体(MAb)作为包被抗体,Eu3+标记的另一株MAb作为检测抗体,建立了犬细小病毒的双抗体夹心时间分辨荧光免疫分析方法,为犬细小病毒的检测提供了一种新的技术手段。结果发现:灵敏度高,约为0.79ng/mL;各浓度CPV标准品在不同水平稀释度回收率为96.48%-105.17%;特异性强,同步检测CCV、CDV、CAV-1、CPIV标准品均为阴性;重复性好,批内和批间变异系数分别在4.78%-6.03%和4.23%-6.42%;稳定性好,4℃可保存6个月以上,37℃可保存7天以上。临床检测结果与PCR方法一致。
本发明提供的检测方法具有灵敏度高,稳定性好,特异性强,重复性好,操作便捷,受本底因素影响少等优点,适用于CPV的快速检测,具有较高的临床实用价值和推广价值。
附图说明
图1 CPV时间分辨免疫荧光技术的标准曲线
具体实施方式
材料来源:
犬细小病毒单克隆抗体4C11(包被抗体,效价1∶150000)和7D5(检测抗体,效价1∶300000)均购买珠海博美生物科技有限公司;
铕(Eu3+)标记试剂盒购自PerkinElmer公司;
牛血清白蛋白(BSA)购自Sigma-Aldrich公司;
Sephadex-G50填料购自GE公司;
微孔板购自Costar公司;
BCA蛋白定量试剂盒购自Sigma-Aldrich公司;
细小病毒抗原快速检测试剂盒为Anigen;
干式荧光免疫分析仪购买于苏州和迈精密仪器有限公司;
60份疑似犬细小病毒的样本,20份犬细小病毒阴性粪便均由上海市农业科学院宠物医院提供。
犬细小病毒(Canine parvovirus,CPV)、犬冠状病毒(Canine coronavirus,CCV)、犬瘟热病毒(canine distemper,CDV)、犬腺病毒I型(Canine adenovirus type I,CAV-I)、犬副流感病毒(Canine parainfluenza virus,CPIV)均由上海市农业科学院畜牧兽医研究所提供。
实施例1检测犬细小病毒TRFIA法的建立和性能评估
1.1固相包被抗体的制备单克隆抗体4C11用2μg/mL包被液(碳酸盐缓冲液,50mmol/L,pH 9.6)包被,每孔100μL,4℃过夜,弃包被液,加封闭液(5%BSA的PBS溶液,pH7.4),每孔250μL,37℃封闭2h,弃封闭液,用洗涤液(0.5%Tween-20的PBS溶液,pH 7.4)洗涤3次,拍干,放于-20℃保存备用。
1.2铕标记检测抗体及其纯化参考铕标记Eu3+试剂盒说明书对检测抗体7D5进行标记。取0.5mg检测抗体7D5,加入到带有滤膜的离心管中,8000×g离心8min。用标记缓冲液(50mmol/L,Na2CO3,pH 9.0)重复洗涤6次。
设置Eu3+标记试剂:标记抗体比例分别为70μL:30μL,60μL:40μL,50μL:50μL,40μL:60μL,30μL:70μL,25℃振荡过夜。标记完成后用Sephadex-G50层析分离纯化,用洗脱液(含50mmol/L Tris-HCL的生理盐水)洗脱,收集流出液(1mL/管),测定每管洗脱液的OD280值,并根据Eu3+标记试剂盒说明书所提供的公式计算标记率。
1.3标准品的制备用含0.02%BSA,0.05%Proclin300,50mmol/L的Tris-HCL缓冲液(pH 7.8),将CPV抗原配制成浓度为0,5ng/mL,10ng/mL,20ng/mL,40ng/mL,80ng/mL,160ng/mL,640ng/mL的系列标准品溶液,采用BCA法测定CPV抗原浓度,每瓶1mL分装冻干,放-20℃保存备用。使用时分别加入1mL去离心水溶解使用。
1.4 TRFIA法检测犬细小病毒步骤取25μL标准品或待测样品,加入到包被好的微孔板中,再加入200μL分析缓冲液(含8mmol/L NaCL,0.1%明胶,0.05%Proclin300,0.1%mL/LTween-80的Tris-HCL溶液,pH7.8),25℃振荡孵育1h,弃掉上清液,用洗涤液洗涤3次,加入150μL Eu3+标记的CPV抗体溶液,再加入100μL分析缓冲液,混均25℃孵育1h,去除上清,洗涤液洗涤5次,加入200μL增强液;处理好的微孔板于干式荧光免疫分析仪上读取荧光值。
TRFIA方法检测性能评估
一、绘制标准曲线及计算灵敏度使用上述配制的CPV标准品(0,5ng/mL,10ng/mL,20ng/mL,40ng/mL,80ng/mL,160ng/mL,640ng/mL)制作标准曲线,每个浓度复测3次,共测定3次,进行线性回归分析及相关系数。以0浓度的CPV抗原标准品为样本,平行测定其20次荧光值,计算均值(mean)和标准差(SD),将其平行测定20次的荧光值均值加上2倍的标准差得到的荧光值(mean+2SD)代入标准曲线方程,计算TRFIA方法的灵敏度。
绘制TRFIA标准曲线并计算灵敏度以CPV标准品浓度为横坐标,相对应的荧光值为纵坐标,绘制标准曲线,如图1所示,曲线方程为y=163.18x+618.79,,R2=0.9996,本检测方法具有良好的计量-反应效应。平行测定10次浓度为0的CPV标准品荧光值,计算其对应的均值(mean)及标准差(SD),mean+2SD带入标准曲线方程,计算得出TRFIA法的检测下限为0.79ng/mL。
二、准确度分析对已知浓度的3个CPV标准品(18.2ng/mL,21.8ng/mL和42.4ng/mL)按照1∶2,1∶4,1∶8倍比稀释,每个样本重复检测3次,比较样本的测定值、真值并计算其稀释回收率(稀释回收率%=测定值/真值×100),通过稀释回收率分析检测方法的准确度。
将3个不同浓度的CPV标准品按1∶2,1∶4和1∶8倍比稀释,用建立的TRFIA方法进行检测。结果显示见表1,三个标准品三个水平稀释度的稀释回收率在96.48%~105.17%。稀释倍数与浓度呈线性关系,表明本研究建立的TRFIA法准确度较高。
表1 CPV TRFIA稀释回收率测定结果
三、特异性分析利用建立的TRFIA方法同时检测浓度为100ng/mL的CPV,CCV,CDV,CAV-1,CPIV标准品,进行特异性实验。
利用建立的TRFIA方法同时检测犬临床常见病毒,浓度均为100ng/mL的CPV、CDV、CCV、CPIV和CAV-1标准品。检测结果为CPV标准品为阳性,其他病毒检测浓度均低于最低检出浓度0.79ng/mL(表2)。表明该方法特异性良好。
表2 CPV TRFIA方法的特异性结果
四、重复性实验将CPV标准品稀释至高(400ng/mL),中(100ng/mL)和低(20ng/mL)3个浓度,分别设定3个复孔,重复检测10次,计算3个浓度标准品测定的均数、标准差(SD)及变异系数(CV),评价其批内重复性;用标准品三个浓度(400ng/mL,100ng/mL和20ng/mL),设定3个复孔,在10个不同时间检测,计算3个浓度标准品测定的均数、标准差及变异系数,评价其批间的重复性。
利用建立的TRFIA方法进行重复性实验,计算批内变异系数在4.78%~6.03%,批间变异系数在4.23%~6.42%(表3)。批内和批间变异系数均小于10%,表明TRFIA方法重复性好。
表3 CPV TRFIA重复性测定结果
统计学方法采用SPSS17.0统计软件。结果以mean±SD表示,Graph Pad Prism5软件计算标准曲线方程,采用Origin8软件进行绘图。
实施例2应用实施例1建立的方法进行样本检测
临床样本检测共收集60份由细小病毒抗原快速检测试剂盒确定为CPV阳性的患病犬的粪便样本(实验样本),20份由细小病毒抗原快速检测试剂盒确定为CPV阴性的粪便样本(对照样本),采用建立的TRFIA方法与PCR法(上游引物序列:CPV-P1:GAATCTGCTACTCAGCCACCAAC;下游引物序列:GTGCACTATAACCAACCTCAGC。PCR反应条件:94℃预变性5min,94℃变性30s,55℃30s,72℃50s,35个循环,72℃延伸5min。)同时检测,对检测结果进行对比分析,来测试TRFIA法的临床检测效果。
利用本方法与PCR方法分别同时检测60份实验样本与20份对照样本。对于60份实验样本,TRFIA方法和PCR方法的检测结果一致,均是58份阳性,2份阴性。20份对照样本检测结果均为阴性。两种检测方法符合率为100%,表明本研究制备的TRFIA能够准确、快速检测CPV临床样本。
Claims (2)
1.一种犬细小病毒的检测方法,其特征在于该方法包括如下步骤:
以单克隆抗体4C11作为包被抗体,铕Eu3+标记的单克隆抗体7D5作为检测抗体,建立了犬细小病毒的双抗体夹心时间分辨荧光免疫分析检测方法。
2.根据权利要求1所述的犬细小病毒的检测方法,包括如下步骤:
(1)固相包被抗体的制备单克隆抗体4C11于微孔板内用2μg/mL包被液,碳酸盐缓冲液,50mmol/L,pH 9.6,包被,每孔100μL,4℃过夜,弃包被液,加封闭液,5%BSA的PBS溶液,pH7.4,每孔250μL,37℃封闭2h,弃封闭液,用洗涤液:0.5%Tween-20的PBS溶液,pH 7.4,洗涤3次,拍干,放于-20℃保存备用;
(2)铕标记检测抗体及其纯化参考铕标记Eu3+试剂盒说明书对检测抗体7D5进行标记。取0.5mg检测抗体7D5,加入到带有滤膜的离心管中,8000×g离心8min;用标记缓冲液,50mmol/L,Na2CO3,pH 9.0,重复洗涤6次;
设置Eu3+标记试剂:标记抗体比例分别为70μL:30μL,60μL:40μL,50μL:50μL,40μL:60μL,30μL:70μL,25℃振荡过夜;标记完成后用Sephadex-G50层析分离纯化,用含50mmol/LTris-HCL的生理盐水洗脱液洗脱,收集流出液,测定每管洗脱液的OD280值,并根据Eu3+标记试剂盒说明书所提供的公式计算标记率;
(3)标准品的制备及标准曲线的绘制用含0.02%BSA,0.05%Proclin300,50mmol/L的Tris-HCL缓冲液,pH 7.8,将CPV抗原配制成浓度为0,5ng/mL,10ng/mL,20ng/mL,40ng/mL,80ng/mL,160ng/mL,640ng/mL的系列标准品溶液,采用BCA法测定CPV抗原浓度,每瓶1mL分装冻干,放-20℃保存备用。使用时分别加入1mL去离子水溶解使用;
(4)检测犬细小病毒取25μL标准品或待测样品,加入到包被好的微孔板中,再加入200μL分析缓冲液,8mmol/L NaCL,0.1%明胶,0.05%Proclin300,0.1%mL/L Tween-80的Tris-HCL溶液,pH 7.8,25℃振荡孵育1h,弃掉上清液,用洗涤液,0.5%Tween-20的PBS溶液,pH7.4,洗涤3次,加入步骤(2)中制备得到的150μL Eu3+标记的CPV抗体溶液,再加入100μL分析缓冲液,分析液含0.2%BSA、0.9%NaCL、19.6mg/L DTPA、0.05%Proclin300,0.1%mL/LTween-20的Tris-HCL 50mmol/L溶液,pH7.8,混均25℃孵育1h,去除上清,洗涤液洗涤5次,加入200μL增强液,0.1%Triton X-100,3.99mg/L β-NTA,19.33mg/LTOPO,1.3g/L邻苯二甲酸氢钾,0.6%冰醋酸,然后检测荧光值。
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