CN112921124B - 一种快速检测病毒的试剂盒 - Google Patents
一种快速检测病毒的试剂盒 Download PDFInfo
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- CN112921124B CN112921124B CN202110376201.1A CN202110376201A CN112921124B CN 112921124 B CN112921124 B CN 112921124B CN 202110376201 A CN202110376201 A CN 202110376201A CN 112921124 B CN112921124 B CN 112921124B
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Abstract
本发明公开了一种快速检测病毒的试剂盒。本发明通过针对寨卡病毒NS1序列进行分析获得了特异性针对NS1检测的RPA引物和探针,并制备成为相应的检测试纸条;同时针对NS1蛋白筛选并获得效果较好的单克隆抗体,将所述单克隆抗体标记量子点和其他抗体标记的硝酸纤维素膜制备获得寨卡病毒荧光量子点快速检测试纸,将两个检测方法进行组合使用,能够进一步提高检测的准确性,并且成本低廉,适于大规模推广使用。
Description
技术领域
本发明涉及病毒诊断领域,并且更具体地涉及一种快速检测病毒的试剂盒。
背景技术
寨卡病毒(Zika virus)是近年兴起一种的与登革热、日本脑炎、西尼罗河热病毒等高度相似的虫媒黄病毒。该病毒是一种单链的RNA病毒,基因组全长11kb,编码3个结构蛋白(C、prM/M和E),及7个非结构蛋白(NS1、NS2A、NS2B、NS3、NS4A、NS4B和NS5)。ZIKV在全球的大规模爆发,导致包括成人格林-巴利综合征和新生胎儿的先天性畸形等严重疾病。
目前检测ZIKV的主要检测方法有病原学检测、血清学检测。其中病原学检测主要为核酸检测(RT-qPCR,LAMP),血清学检测包括:血清特异性IgM抗体(ELISA、IFA)和中和抗体。病原学检测精准性高、检测时间短,但是设备昂贵,易出现假阳性。此外,由于病毒出现的窗口期短,基于核酸的检测方法很难准确检出。血清学检测简便、快速,但是IgM/IgG通常在发病后7天后才能检出,且受不同感染患者的症状发作影响。
非结构蛋白1(Nonstructural protein 1)作为病毒唯一分泌并与宿主相互作用的重要蛋白,在病毒感染、复制及免疫逃逸过程中起着重要作用。NS1蛋白在细胞内形成同源二聚体,与胞内膜系统的脂类结合,参与病毒复制,并以可溶性的形式分泌于细胞外。此外,NS1蛋白作为抗原,可诱导机体产生抗体,是病毒早期诊断的重要标志物。因此,开发基于ZIKV-NS1的检测试剂盒,对ZIKV的临床诊断和治疗具有重要实际应用价值。但NS1核酸检测精准性高但病毒出现的窗口期短,易出现假阳性;而NS1在病毒血症阶段可同时检测到,并且在病毒血症阶段后仍可检测到,提供比病毒RNA更长,但黄病毒间交叉反应常见,寨卡病毒易与登革病毒、黄热病毒、圣路易斯脑炎病毒等存在血清学交叉反应,难以鉴别。
发明内容
基于上述发现,本发明克服现有技术的缺陷,提供了一种制备简单、成本低廉、使用方便、不需要高精尖仪器的检测试剂盒。所述试剂盒通过核酸-抗体双重检测方法对寨卡病毒进行检测,进一步提高检测的准确性。
本发明提供以下技术方案:
本发明提供一种用于快速检测寨卡病毒的试纸条RPA(LFD RPA)检测试剂盒,含有侧流层析试纸条(Hybridetect 2T,Milenia Biotec GmbH,Germany),以及有一对引物和一条探针,其中所述上游引物序列如SEQ ID NO:2所示,所述下游引物序列如SEQ ID NO:3所示,所述探针的序列如SEQ ID NO:4所示。
具体的,上游引物(SEQ ID NO:2):
GAGCTCAATGCAATCCTGGAAGAGAATGGAG
下游引物(SEQ ID NO:3):
Biotin-GTCACCATCCACGACAAAGCTGTTATTTGTC
探针序列(SEQ ID NO:4):
FAM-CATGTGGAGAGGTCCACAGAGATTGCCCGTGCCTGTGAACGAGCTGC,
在一些实施例中,本发明的探针在中间距5’端35bp的位置处采用dSpacer修饰,dSpacer分子两侧的距5’端34bp和36bp位置的胸腺嘧啶(dT)分别被荧光基团FAM和淬灭基团BHQ1取代,并且在探针的3’末端通过阻塞基团C3Spacer修饰。
在一些实施例中,如上所述的核酸探针,所述荧光基团可替换为TAMARA,所述淬灭基团可替换为BHQ2;所述的脱碱基位点可替换为四氢呋喃;所述探针3’端的C3Spacer修饰可替换为磷酸化设计或连接biotin-TEG。
所述试纸条带有检测线,检测线上固定有分子A;
序列如SEQ ID NO.3所述的引物,其末端带有特异性结合前述分子A的分子B。所述分子A是生物素配体,分子B是生物素。
在本发明所述的试纸条RPA检测试剂盒中,优选的,所述试剂盒中还包括水解缓冲液,醋酸镁以及ddH2O。
本发明提供的试纸条法,RPA探针中间位置的两个胸腺嘧啶核苷酸上分别标记一个荧光基团和一个荧光淬灭基团,两个基团之间设计一个脱碱基位点(dSpacer),该位点可被具有3'-5'外切酶活性的核酸外切酶III识别、切割,游离荧光基团,从而发出荧光信号随后被荧光检测的仪器;同时留下可延伸3’-OH,DNA聚合酶以探针为“正向引物”继续延伸合成DNA,与反向引物(带亲和标记,例如生物素)一起扩增出一种带有双重标记(荧光基团标记、亲和标记)的扩增产物;该产物在侧向流检测试纸中层析,遇到可识别亲和标记的试纸区域(通常为一条线,即“检测线”,带有链霉亲和素)时,则被富集,表现出线形荧光信号。试纸条法由于不依赖荧光定量PCR仪,成本和适用范围更广。
本发明进一步的,提供一种非疾病诊断目的的寨卡病毒检测方法,它是使用前述引物和探针对样品进行扩增,再用核酸检测试纸条检测扩增产物即可。结果检测:结合试纸条进行显色,上述扩增产物吸取5μL-25μL吸取用缓冲液1×PBST稀释10倍-50倍,用相应标记的试纸条进行检测。结果判读:同时出现T线和C线为阳性(+),仅出现C线为阴性(-),只出现T线需考虑试纸条是否有效性。
本发明另外提供一种特异性结合ZIKV-NS1蛋白的单克隆抗体ZS-1701,其包含重链可变区和轻链可变区,该重链可变区含有CDR1区、CDR2区和CDR3区,重链CDR1区、CDR2区和CDR3区的氨基酸序列分别如SEQ ID NO:7、8和9所示;该轻链可变区含有CDR1区、CDR2区和CDR3区,其中轻链CDR1区、CDR2区和CDR3区的氨基酸序列分别如SEQ ID NO:10、11和12所示。
另一方面,本发明提供一种特异性结合NS1蛋白的单克隆抗体ZS-1701,其包含重链可变区和轻链可变区,其中重链可变区包含氨基酸序列SEQ ID NO:5,轻链可变区包含氨基酸序列SEQ ID NO:6。
在一些实施方案中,本发明所述的抗NS1抗体包含两条重链和两条轻链,或由两条重链和两条轻链构成,其中各重链包含上述的重链恒定区序列、重链可变区序列或CDR序列,且各轻链包含上述的轻链恒定区序列、轻链可变区序列或CDR序列。本发明的抗体可以是全长抗体,所述全长抗体包含恒定区,所述全长抗体轻链恒定区进一步包含鼠源κ、λ链序列。所述全长抗体重链恒定区进一步包含鼠源IgG1、IgG2a、IgG2b、IgG3、IgA或IgM序列。
在一些实施方式中,本发明的抗NS1抗体是由上述的抗NS1抗体或抗体片段形成的Fab片段、Fab'片段、F(ab')2片段、Fv片段、双抗体、线性抗体、单链抗体分子或多特异性抗体。
本发明另外提供一种寨卡病毒荧光量子点快速检测试纸,其中将ZS-1701单克隆抗体标记量子点配制备而成。
在一些实施方式中,本发明提供一种核酸-抗体双重检测寨卡病毒的试剂盒,所述试剂盒含有特异性检测NS1核酸的RPA试剂盒以及特异性检测NS1蛋白的含有单克隆抗体的试纸条;所述RPA试剂盒含有SEQ ID NO:2和3的引物以及SEQ ID NO:4的探针;所述单克隆抗体的试纸条为将抗NS1单克隆抗体ZS-1701标记量子点制备而成的荧光量子点快速检测试纸条;其中,所述抗NS1单克隆抗体ZS-1701包括重链可变区,其包含SEQ ID NO:7所示的CDR1、SEQ ID NO:8所示的CDR2和SEQ ID NO:9所示的CDR3,以及轻链可变区,其包含SEQ IDNO:10所示的CDR1、SEQ ID NO:11所示的CDR2和SEQ ID NO:12所示的CDR3。
在一些实施方式中,本发明提供一种核酸-抗体双重检测寨卡病毒的试剂盒,所述试剂盒含有特异性检测NS1核酸的RPA试剂盒以及特异性检测NS1蛋白的含有单克隆抗体的试纸条;所述RPA试剂盒含有SEQ ID NO:2和3的引物以及SEQ ID NO:4的探针;所述单克隆抗体的试纸条为将抗NS1单克隆抗体ZS-1701标记量子点制备而成的荧光量子点快速检测试纸条;其中,所述抗NS1单克隆抗体ZS-1701单克隆抗体的重链可变区序列如SEQ ID NO:5所示,轻链可变区序列如SEQ ID NO:6所示。
一种检测寨卡病毒试剂盒的应用,所述试剂盒通过体外测定来自受试者的生物试样中的NS1蛋白质、或编码该蛋白质的基因的表达。
在一些实施方式中,本发明所述的生物试样为血液、血浆、血清、尿或细胞。
有益效果
本发明通过针对NS1核酸序列进行分析获得了特异性针对NS1核酸的RPA引物和探针,并制备出NS1核酸检测试剂;同时针对NS1抗原筛选并获得效果较好的单克隆抗体,将所述单克隆抗体标记量子点和其他抗体标记的硝酸纤维素膜制备获得寨卡病毒荧光量子点快速检测试纸,将以上两个检测方法进行组合使用,用于寨卡病毒患者的诊断,能够进一步提高检测的准确性,并且成本低廉,适于大规模推广使用。
附图说明
附图用来提供对本发明的进一步理解,并且构成说明书的一部分,与本发明的实施例一起用于解释本发明,并不构成对本发明的限制。
图1RPA检测方法灵敏度评价结果图。
图2小鼠抗体亚型鉴定结果
具体实施方式
以下结合附图对本发明的优选实施例进行说明,应当理解,此处所描述的优选实施例仅用于说明和解释本发明,并不用于限定本发明。
实施例1.RPA特异性检测引物的设计
对寨卡病毒常见株的基因序列进行比对,选择特异性的保守区,作为引物检测的靶标区,其序列SEQ ID NO:1。
根据RPA引物设计的规则并对引物进行优化,获得了特异性的引物序列,其序列如下所示:
上游引物(SEQ ID NO:2):
GAGCTCAATGCAATCCTGGAAGAGAATGGAG
下游引物(SEQ ID NO:3):
Biotin-GTCACCATCCACGACAAAGCTGTTATTTGTC
探针序列(SEQ ID NO:4):
FAM-CATGTGGAGAGGTCCACAGAGATTGCCCGTGCCTGTGAACGAGCTGC,
在一些实施例中,本发明的探针在中间距5’端35bp的位置处采用dSpacer修饰,dSpacer分子两侧的距5’端34bp和36bp位置的胸腺嘧啶(dT)分别被荧光基团FAM和淬灭基团BHQ1取代,并且在探针的3’末端通过阻塞基团C3Spacer修饰。
实施例2.RPA反应灵敏度检测
以寨卡病毒裂解液提取RNA作为模板,进行RPA试验,采用筛选的引物进行RPA扩增,同时设置超纯水为阴性对照,反应温度为38℃,反应时长为20min,RPA反应体系为50μl,其中,2μl正反向引物(10μM),2μl反向引物(10μM),0.6μl探针,25μl含重组酶、DNA聚合酶、单链结合蛋白、核酸内切酶IV、逆转录酶的缓冲液,1μl模板和17.9μlddH2O,充分振荡混匀并且瞬离,最后加入2.5μl的280mM醋酸镁(MgOAc),将反应管置于实时荧光PCR仪中38℃恒温反应相应时长;结果如图1所示。如图1所示,当模板浓度为1ng,100pg,10pg时,均出现明显扩增曲线与目的条带,但模板浓度低于1pg时无明显扩增曲线与目的条带,即说明RPA的检测最低限为10pg,具有较好的检测精度。
实施例3.RPA反应特异性检测
在进行试纸条RPA(LFD-RPA)的反应特异性的检测时,按照上述体系加入反应物。使用模板分别为寨卡病毒(Zika virus,ZIKV)、登革病毒(Dengue virus,DENV)、黄热病毒(Yellow fever virus,YFV)。RPA反应体系为50μl,其中,2μl正反向引物(10μM),2μl反向引物(10μM),0.6μl探针,25μl含重组酶、DNA聚合酶、单链结合蛋白、核酸内切酶IV、逆转录酶的缓冲液,1μl模板和17.9μlddH2O,充分振荡混匀并且瞬离,最后加入2.5μl的280mM醋酸镁(MgOAc)。反应温度设为38℃的水浴锅中进行,反应时间设定为20min。结果表明,只有寨卡病毒样本同时出现T线和C线为阳性(+),其余样本仅出现C线为阳性(+),说明本发明的试纸条具有很好的特异性,结果如下表1所示。
表1
病毒株 | LFD RPA T线 | LFD RPA C线 |
寨卡病毒 | + | + |
登革病毒 | - | + |
黄热病毒 | - | + |
实施例4.NS1抗原制备
将ZIKV-NS1蛋白对应的DNA片段通过XbaI和XhoI酶切后,连接到pET21a载体上。其中ZIKV-NS1蛋白编码区的3’端连上6个组氨酸标签(6xHis-tag)的编码序列及翻译终止密码子。再将连接产物转化到BL21大肠杆菌感受态细胞。单克隆接种到40Ml LB培养基中,培养6-8小时。接种到4L的LB培养基中,37℃培养至OD600=0.4-0.6,加入IPTG至终浓度1mM,37℃继续培养4-6小时。收获包涵体,通过稀释法复性包涵体。复性液浓缩后换成20mMTris,150mM NaCl,pH8.0缓冲液。将浓缩后的蛋白溶液进一步以分子排阻层析纯化,使用AKTA-purifier(GE)和superdex200Hiload 16/60柱子(GE),使用缓冲液A(20mM Tris,150mM NaCl,pH8.0),同时监测280nm的紫外吸收值,收取目的蛋白。使用BCA蛋白定量试剂盒测定重组蛋白浓度(购自上海朝瑞生物科技有限公司)。
实施例5.抗NS1抗体制备
用PBS将经纯化处理的蛋白稀释至0.5mg/ml后,将其与弗氏完全佐剂按1:1的比例混合乳化,皮下多点免疫4只6~8周龄的雄性BALB/c小鼠,剂量为400μl/只。将稀释的蛋白与弗氏不完全佐剂按1:1的比例混合乳化,间隔两周后用相同的剂量,小鼠皮下多点注射加强免疫,两周一次,一共免疫两次。每次都在免疫的前一天采取小鼠的眼球静脉血,间接ELISA检测血清中抗体的滴度变化。在细胞融合前3天,对小鼠进行脾脏加强免疫,剂量为200μl/只。于第四天取脾,进行细胞融合。取免疫后的Balb/c小鼠脾细胞,将其与骨髓瘤Sp2/0细胞株融合,将融合后的细胞用20%FBS-HAT-DMEM培养基重悬,然后均匀铺于96孔板内,37℃,5%CO2培养。融合后的细胞在培养一周左右后用10%FBS-HT-DMEM培养基进行半量换液,当细胞菌落覆盖孔底的面积达到1/3~1/2时,取培养上清,用间接ELISA方法进行阳性克隆的检测。选取OD450nm值较高且细胞集落数目少的孔,通过有限稀释法进行克隆化。同样以ELISA方法进行筛选,最终获得阳性杂交瘤细胞株并将其命名为ZS-1701。扩大培养后,冻存杂交瘤细胞。
实施例6.单克隆抗体的纯化
取BALB/c小鼠,在接种杂交瘤细胞前1周,经腹腔注射降植烷,0.5ml/只。1周后,每只小鼠经腹腔接种约1X106个杂交瘤细胞;7~10d后,收集腹水。将腹水经10000×g离心30min后,弃沉淀,用50%硫酸铵盐析粗提,PBS溶解,流水透析5h;用0.1mol/L磷酸盐缓冲液(pH8.0)透析平衡过夜;上样,用0.1mol/L磷酸盐缓冲液(pH8.0)洗脱杂蛋白,用不同pH值的柠檬酸盐洗脱液洗脱,分段收集洗脱峰,浓缩后,得到纯化后的抗NS1抗体ZS-1701。
实施例7.抗NS1抗体亚型鉴定
根据亚类测定试剂(sigma公司)对间接ELISA筛选出的阳性鼠单克隆细胞株进行亚类测定。试剂盒中提供的酶标板上已经预包被了针对小鼠IgG1、IgG2a、IgG2b、IgG3、IgA、IgM、kappa轻链、lambda轻链的特异性抗体,将实施例6中纯化的抗NS1抗体ZS-1701样品加入样品孔中,每孔50μl,无需孵育。将1X羊抗鼠IgA+IgM+IgG-HRP加入样品孔中,每孔50μl,轻轻混匀后,孵育1h。扣去孔中液体加入1XPBST洗孔3次,吸水纸吸干多余水分。加入显色液,每孔100μl,室温避光显色15min。加入100μl终止液,终止显色反应。结果如图2所示,本发明的单克隆抗体为IgG1亚型。
实施例8.单克隆抗体序列确定
从液氮中取出ZS-1701杂交瘤细胞细胞冻存管,于37℃快速融解,1000rpm,5min离心去除冻存液,置于100mm孔板,培养至占培养板约80%,加入1ml Trizol试剂(Thermo公司),并依据说明书提取杂交瘤细胞总RNA。取2.5μg上述总RNA,加入DECP水,使体积达到11μl,加入1.0μl oligo(dT)(10μM),加入1μl dNTPs(10mM),混合均匀,65℃孵育5分钟后置冰上1分钟,然后加入4μl RT buffer(5×),1.0μl DTT(100mM),1μl RibonucleaseInhibitor及1μl逆转录酶(takara公司),50℃反应10min。80℃孵育10分钟以终止反应,获得的cDNA保存在-20℃。设计特异性的巢式PCR引物,该扩增反应中所使用的引物序列与抗体可变区第一框架区和恒定区互补,采用常规PCR方法扩增目的基因。其中,引物序列的设计按照文献(Bodo Brocks.Species-Crossreactive scFv Against the Tumor StromaMarker“Fibroblast Activation Protein”Selected by Phage Display From anImmunized FAP-/-Knock-Out Mouse)进行。
测序结果显示,本发明所述的抗NS1抗体ZS-1701的重链和轻链可变区的氨基酸序列分别如SEQ ID NO:5和SEQ ID NO:6所示;该抗体的重链可变区3个CDR的氨基酸序列分别如SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9所示;轻链可变区3个CDR的氨基酸序列分别如SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12所示。
实施例9.抗NS1抗体亲和力测定
将抗体捕获抗体(AHC)通过氨基偶联的方式包被在CM5芯片表面,参照氨基偶联试剂盒和抗捕获试剂盒说明书,准备芯片活化缓冲液盐酸N-乙基-N’-(3-二甲氨基丙基)-碳二亚胺(EDC)和N-羟基琥珀酰亚胺(NHS)、AHC和封闭用的乙醇胺,选择Biacore3000系统中的包被程序,将AHC氨基偶联在CM5芯片表面。将实施例6中获得的抗NS1抗体ZS-1701捕获于芯片表面。用HBS-EP+缓冲液将抗体稀释至1μg/mL,设置流速为10μl/min,包被至响应值达100RU。NS1抗原设置7个不同浓度梯度,用HBS-EP缓冲液依次进行2倍梯度稀释,浓度从40nM到0nM。设置NS1抗原的流速为30μl/min,结合时间为3min。设置HBS-EP+缓冲液流速为30μl/min,解离时间为10min。使用3M MgCl2作为再生缓冲液,按照再生程序对芯片进行再生。通过同时拟合结合和解离传感图计算结合速率(Ka)和解离速率(Kd)。平衡解离常数(Kd)用解离速率(Kd)/结合速率(Ka)计算。结果如表2所示:本发明的抗体亲和力较高,亲和力KD值达到5.43x10-8M。
表2
抗体 | K<sub>a</sub>(1/Ms) | K<sub>d</sub>(1/s) | K<sub>D</sub>(M) |
ZS-1701 | 2.78x10<sup>4</sup> | 1.51x10-<sup>3</sup> | 5.43x10-<sup>8</sup> |
实施例10.ZIKV-NS1多克隆抗体的制备
取实施例4中制备的NS1抗原,按照每只兔子1mg蛋白的量进行免疫。将1mg蛋白溶于500μl无菌PBS中,然后加入等体积的佐剂,在漩涡震荡器上混匀1h,然后用注射器吹吸混匀后皮下多点注射。14天后重复该实验,多次免疫后,采取耳缘静脉血进行抗血清效价的检测。当抗血清的效价达到融合标准,进行一次加强免疫后,选用一次全采血法,自颈动脉无菌放血,分离血清,加入适量防腐剂,分装小瓶,低温冰箱保存。用50%硫酸铵沉淀一次,33%硫酸铵沉淀两次,然后用DEAE层析法把兔血清进行纯化,提取IgG。用间接ELISA法测定纯化的IgG效价,得到抗ZIKV-NS1多克隆抗体。
实施例11.寨卡病毒荧光量子点快速检测试纸的制备及验证
将实施例6纯化的ZS-1701单克隆抗体标记量子点,实施例10纯化的ZIKV-NS1多克隆抗体包被硝酸纤维素膜搭配检测。将其制备成快速检测试纸后,分别对其检出限、交叉反应进行测试。将国家参考品中的最低检出限样品S1,寨卡病毒(病毒滴度1×106TCID50/L),用0.02mol/L PBS缓冲液进行1∶10、1∶20、1∶40、1∶80、1∶160、1∶320、1∶640、1∶1280、1∶2560倍稀释后检测,检测结果见表3,在365nm紫外灯照射下,样品在1∶640倍稀释后,试纸检测线仍然可见荧光条带,因此寨卡病毒荧光量子点快速检测试纸的检出限为1.56×103TCID50/L。
表3
稀释倍数 | 结果 |
1∶10 | + |
1∶20 | + |
1∶40 | + |
1∶80 | + |
1∶160 | + |
1∶320 | + |
1∶640 | + |
1∶1280 | - |
1∶2560 | - |
同时将所述寨卡病毒荧光量子点快速检测试纸检测登革病毒(Dengue virus,DENV)、黄热病毒(Yellow fever virus,YFV),西尼罗病毒(West Nile virus,WNV)、日本脑炎病毒(Japanese encephalitis virus,JEV),观察是否存在交叉反应,检测结果表明寨卡病毒荧光量子点快速检测试纸与所测样品没有交叉反应,具有良好的特异性。
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Claims (1)
1.一种核酸-抗体双重检测寨卡病毒的试剂盒,所述试剂盒含有试纸条RPA检测试剂盒和抗体检测试剂盒,所述试纸条RPA检测试剂盒包括一对引物和一条探针,所述的一对引物的序列如SEQ ID NO:2和3所示,所述探针的序列如SEQ ID NO:4所示;所述抗体检测试剂盒含有一种特异性结合寨卡病毒NS-1抗原的抗体,所述抗体的重链可变区序列如SEQ ID NO:5所示,轻链可变区序列如SEQ ID NO:6所示。
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Citations (16)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN106478815A (zh) * | 2016-10-19 | 2017-03-08 | 广州市第八人民医院 | 快速制备寨卡病毒特异全人源单克隆抗体的方法与应用 |
CN106497884A (zh) * | 2016-10-28 | 2017-03-15 | 南方医科大学 | 一种重组hek‑293t细胞及其分泌寨卡病毒非结构蛋白1 |
WO2017132210A1 (en) * | 2016-01-25 | 2017-08-03 | Iogenetics, Llc | Interventions for flavivirus infections |
CN107102144A (zh) * | 2017-03-16 | 2017-08-29 | 深圳市梓健生物科技有限公司 | 快速检测寨卡病毒ns1蛋白的荧光定量免疫层析试纸条及其制作方法 |
WO2018115509A3 (en) * | 2016-12-23 | 2018-08-30 | Expres2Ion Biotechnologies Aps | New flavivirus vaccine |
CN107188935B (zh) * | 2017-07-20 | 2018-10-02 | 北京健乃喜生物技术有限公司 | 一种寨卡病毒ns1抗原突变体及其应用 |
CN109071610A (zh) * | 2016-05-03 | 2018-12-21 | 无微华斯生物科技有限公司 | 用于诊断和治疗病毒感染的方法和系统 |
CN109406788A (zh) * | 2018-11-13 | 2019-03-01 | 昆明理工大学 | 一种单克隆抗体及其应用 |
CN109503712A (zh) * | 2018-12-12 | 2019-03-22 | 广州市第八人民医院 | 一种单克隆抗体ZKns2E11及其应用 |
CN109563157A (zh) * | 2016-07-13 | 2019-04-02 | 胡默波斯生物医学公司 | 特异性地结合至寨卡病毒表位的新型抗体和其用途 |
CN110387435A (zh) * | 2018-04-17 | 2019-10-29 | 中国人民解放军军事科学院军事医学研究院 | 利用rpa技术检测寨卡病毒的方法及其专用成套试剂 |
CN110441521A (zh) * | 2018-05-03 | 2019-11-12 | 深圳市疾病预防控制中心 | 检测Zika病毒NS1抗原的试纸条和试剂盒及方法 |
CN110568178A (zh) * | 2019-09-18 | 2019-12-13 | 中国检验检疫科学研究院 | 一种寨卡病毒ns1抗原及其在制备荧光免疫层析试剂中的应用 |
CN110699491A (zh) * | 2019-11-13 | 2020-01-17 | 中国疾病预防控制中心病毒病预防控制所 | 寨卡病毒实时荧光定量rt-rpa检测引物和探针及检测试剂盒 |
CN110724767A (zh) * | 2019-11-13 | 2020-01-24 | 中国疾病预防控制中心病毒病预防控制所 | 用于检测登革热、基孔肯雅及寨卡病毒的三重实时荧光定量pcr引物和探针及试剂盒 |
WO2020092564A1 (en) * | 2018-10-31 | 2020-05-07 | Icahn School Of Medicine At Mount Sinai | Human antibodies targeting zika virus ns1, ns1 polypeptides and uses thereof |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN112280901B (zh) * | 2020-11-11 | 2021-05-14 | 浙江恒驭生物科技有限公司 | 一种改进的核酸检测技术在制备病毒检测试剂盒中的用途 |
CN112063765B (zh) * | 2020-11-11 | 2021-03-12 | 广东赛尔生物科技有限公司 | 一种核酸抗体双重检测病毒的试剂盒及其制备方法 |
-
2021
- 2021-04-08 CN CN202110376201.1A patent/CN112921124B/zh not_active Expired - Fee Related
Patent Citations (16)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2017132210A1 (en) * | 2016-01-25 | 2017-08-03 | Iogenetics, Llc | Interventions for flavivirus infections |
CN109071610A (zh) * | 2016-05-03 | 2018-12-21 | 无微华斯生物科技有限公司 | 用于诊断和治疗病毒感染的方法和系统 |
CN109563157A (zh) * | 2016-07-13 | 2019-04-02 | 胡默波斯生物医学公司 | 特异性地结合至寨卡病毒表位的新型抗体和其用途 |
CN106478815A (zh) * | 2016-10-19 | 2017-03-08 | 广州市第八人民医院 | 快速制备寨卡病毒特异全人源单克隆抗体的方法与应用 |
CN106497884A (zh) * | 2016-10-28 | 2017-03-15 | 南方医科大学 | 一种重组hek‑293t细胞及其分泌寨卡病毒非结构蛋白1 |
WO2018115509A3 (en) * | 2016-12-23 | 2018-08-30 | Expres2Ion Biotechnologies Aps | New flavivirus vaccine |
CN107102144A (zh) * | 2017-03-16 | 2017-08-29 | 深圳市梓健生物科技有限公司 | 快速检测寨卡病毒ns1蛋白的荧光定量免疫层析试纸条及其制作方法 |
CN107188935B (zh) * | 2017-07-20 | 2018-10-02 | 北京健乃喜生物技术有限公司 | 一种寨卡病毒ns1抗原突变体及其应用 |
CN110387435A (zh) * | 2018-04-17 | 2019-10-29 | 中国人民解放军军事科学院军事医学研究院 | 利用rpa技术检测寨卡病毒的方法及其专用成套试剂 |
CN110441521A (zh) * | 2018-05-03 | 2019-11-12 | 深圳市疾病预防控制中心 | 检测Zika病毒NS1抗原的试纸条和试剂盒及方法 |
WO2020092564A1 (en) * | 2018-10-31 | 2020-05-07 | Icahn School Of Medicine At Mount Sinai | Human antibodies targeting zika virus ns1, ns1 polypeptides and uses thereof |
CN109406788A (zh) * | 2018-11-13 | 2019-03-01 | 昆明理工大学 | 一种单克隆抗体及其应用 |
CN109503712A (zh) * | 2018-12-12 | 2019-03-22 | 广州市第八人民医院 | 一种单克隆抗体ZKns2E11及其应用 |
CN110568178A (zh) * | 2019-09-18 | 2019-12-13 | 中国检验检疫科学研究院 | 一种寨卡病毒ns1抗原及其在制备荧光免疫层析试剂中的应用 |
CN110699491A (zh) * | 2019-11-13 | 2020-01-17 | 中国疾病预防控制中心病毒病预防控制所 | 寨卡病毒实时荧光定量rt-rpa检测引物和探针及检测试剂盒 |
CN110724767A (zh) * | 2019-11-13 | 2020-01-24 | 中国疾病预防控制中心病毒病预防控制所 | 用于检测登革热、基孔肯雅及寨卡病毒的三重实时荧光定量pcr引物和探针及试剂盒 |
Non-Patent Citations (4)
Title |
---|
External Quality Assessment for Molecular Diagnostics of Zika Virus:Experiences from and International EQA Programme,2016-2018;Oliver Donoso Mantke等;《VIRUSES》;20181213;第10卷(第9期);第491页 * |
Innovative and New Approaches to Laboratory Diagnosis of Zika and Dengue : A Meeting Report;Adriana Goncalves等;《Infectious Diseases》;20171225;第217卷(第7期);第1060-1068页 * |
寨卡病毒NS1蛋白的原核表达和单克隆抗体的制备;赵亭亭等;《中国人兽共患病学报》;20190122;第35卷(第03期);第196页摘要,第199页右栏2.4部分 * |
赵亭亭等.寨卡病毒NS1蛋白的原核表达和单克隆抗体的制备.《中国人兽共患病学报》.2019,第35卷(第03期), * |
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