CN117887896A - 一种肠道病毒通用和分型rt-pcr检测试剂盒及假病毒制备方法 - Google Patents
一种肠道病毒通用和分型rt-pcr检测试剂盒及假病毒制备方法 Download PDFInfo
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Abstract
本发明提供了一种肠道病毒通用和分型RT‑PCR检测试剂盒及假病毒制备方法;所述的检测试剂盒同时包括互不干扰和互不影响的肠道病毒通用EV和EVA71、CVA6、CVA16的Q‑PCR分型引物对和特异性的探针,能够实现一步同时准确检测肠道病毒EV和EVA71、CVA6、CVA16的分型;假病毒制备将编码EVA71、柯萨奇病毒CVA6、CVA16衣壳蛋白的VP1基因连入病毒载体,转染细胞,收集假病毒颗粒,纯化假病毒颗粒,最终用保护剂溶解分装;检测试剂盒通过一步法荧光定量PCR进行检测,操作简便,能够达到快速、准确筛查的效果,为EV的早期诊断和治疗提供科学依据;制备的假病毒无致病性,制备效率高,可冻干可长期稳定大量制备,为后续临床广泛应用提供良好基础。
Description
技术领域
本发明属于肠道病毒检测技术领域,具体涉及一种肠道病毒EV通用和柯萨奇病毒A6、16型、肠道病毒71型分型的RT-PCR检测试剂盒及假病毒的制备方法。
背景技术
人类肠道病毒(Human Enteroviruses,HEVs)属于小RNA病毒科(Picornaviridae)肠道病毒属(Enterovirus)。根据HEVs抗原性及致病性的不同,被分为至少72个血清型:人脊髓灰质炎病毒、人柯萨奇病毒(Coxsackievirus,CV)A组、B组、埃可病毒(Echovirus,Echo)和新型肠道病毒。手足口病是由多种肠道病毒感染引起急性传染病,以柯萨奇病毒A16型和EV71为最常见类型。常见于6岁以下婴幼儿,引起手、足、口等部位皮疹疱疹、咽颊炎、心肌炎、脑炎,严重者可导致死亡。
手足口病的流性特征逐渐转变为CVA16、CVA6、CVA10等柯萨奇病毒占主导,多种肠道病毒共存。目前,肠道病毒的诊断方法有病毒分离培养、血清学实验、核酸分子检测。病毒分离培养是确诊的金标准,但其操作繁琐,时间长,且阳性率较低,不利于疾病诊断、监测和治疗。因此,多重实时荧光定量PCR技术封闭式反应、污染少、简便快捷、重复性好等优势,克服了以往临床诊断的缺陷,具有较高的特异性和灵敏性,为临床诊断提供了准确可靠的实验室依据,可作为一种临床病原诊断的有效、快速的检测手段。
现有技术表明病毒核酸PCR检测应用的质控品;例如cDNA、质粒DNA、裸露的RNA以及体外转录的RNA、灭活的病毒颗粒,都难以达到试验要求需要研制稳定的、无生物传染性的质控物和标准品,不但对RNA病毒的RT-PCR检测,而且对商品化的病毒RT-PCR试剂盒的评价都具有重要意义。由于假病毒的技术不断成熟发展,在体外通过特殊方法将病毒样颗粒与重组质粒DNA融合,转录成的重组RNA包入病毒样颗粒内,即形成假病毒(pseudovirus)。
本发明提供了一种能够同时检出肠道病毒的存在,并进行主要肠道病毒EV71、CVA16和CVA6病毒分型检测的方法,比单重荧光定量PCR方法更便捷迅速、节约成本。另外,提供了一种假病毒制备的方法能够携带肠道病毒外源病毒的特定基因片段,模拟真病毒结构、能够抵抗核酸酶降解、稳定、安全性高等优点,给目前RNA病毒检测研究工作提供了一种强有力的工具。
发明内容
本发明的目的在于:提供了肠道病毒EV通用和柯萨奇病毒A6、16型、肠道病毒71型分型的RT-PCR检测试剂盒及假病毒的制备方法,能够一步同时检测肠道病毒EV,并进行柯萨奇病毒A6、16型、肠道病毒71的分型鉴定,引物对之间互不干扰,检测灵敏度高,提升检测效率,满足多种需求。
本发明另一个目的在于:提供了一种假病毒制备的方法,该假病毒能够携带肠道病毒的特定基因片段,模拟真病毒结构无复制活性,安全性较高。
本发明的技术方案如下:
一种肠道病毒通用和分型RT-PCR检测试剂盒,包括通用EV、EVA71、CVA6、CVA16的特异性引物、探针组混合液,酶混合液,阳性对照品,阴性对照品组成。
根据一种优选的实施方式,所述的EVA71特异性引物、探针组如SEQ ID NO:2~SEQID NO:4所示的引物1、2和探针1;
所述的CVA6特异性引物、探针组如SEQ ID NO:6~SEQ ID NO:8所示的引物3、4和探针2;
所述的CVA16特异性引物、探针组如SEQ ID NO:10~SEQ ID NO:12所示的引物5、6和探针3;
所述通用的EV特异性引物、探针组如SEQ ID NO:14~SEQ ID NO:16所示的引物7、8和探针4。
优选地,所述探针1标记FAM荧光,所述探针2标记HEX荧光,所述探针3标记ROX荧光,所述探针4标记CY5.5荧光;通过四重实时荧光同时检测出样本中的肠道病毒EV以及常见的EVA71、CVA6、CVA16病毒分型。本发明产品适用于临床病原的快速、有效、的诊断手段,帮助医生快速判断病人是否感染肠道病毒,以便后续的精准治疗。
一种肠道病毒通用和分型RT-PCR检测试剂盒的应用方法,包括如下步骤:将样品核酸加入所述试剂盒,进行实时荧光PCR;若检测结果Ct≤35,且有明显指数增长期,则为阳性;反之,则为阴性。
优选的,实时荧光定量PCR的反应条件为:反转录50℃、15min、1个循环;预变性95℃、10min;变性94℃、15s,退火延伸58℃、40s,40个循环采集荧光。
一种肠道病毒假病毒的制备方法为:将肠道病毒EVA71、柯萨奇病毒CVA6、CVA16的VP1基因分别连入慢病毒载体,转染293T细胞,收集假病毒颗粒,纯化假病毒颗粒。
优选的,所述的编码EVA71衣壳蛋白的VP1基因核苷酸序列如SEQ ID No.1所示;
CVA6衣壳蛋白的VP1基因核苷酸序列如SEQ ID No.5所示;
CVA16衣壳蛋白的VP1基因核苷酸序列如SEQ ID No.9所示.
进一步的,肠道病毒假病毒的制备方法具体包括以下步骤:
(1)将GeneBank中已经的收录的VP1基因完整翻译蛋白片段5’端、3’端添加相应酶切位点和Flag标签,与慢病毒载体pLVX-Puro连接合成目的质粒;
(2)将步骤(1)所述的目的基因质粒转染到293T细胞48h后,收集细胞并将其裂解,提取蛋白样本,Western blot验证质粒表达;
(3)将所述目的质粒、包装质粒pCMV-dR8.74、包膜质粒pMD2.G混合、转染293T细胞后获得重组假病毒。
(4)将步骤(3)所述假病毒利用PEG进行浓缩纯化,制备假病毒。
优选的,步骤(3)的中所述的转染时所需质粒量为6ug,即包膜质粒pMD2.G、包装质粒pCMV-dR8.74、目的质粒×flag的质量比1:2:3。
优选的,步骤(3)、步骤(4)中,所述的假病毒制备,将转染后293T细胞,在含10%FBS的DMEM培养基中在37℃、5%CO2条件下继续培养48h,培养结束后收取上清液,经离心、过滤后得到所述假病毒。
本发明的实施,至少具有以下优势:
1、一种肠道病毒通用和分型RT-PCR检测试剂盒,应用方便:研发出了能够同时检测肠道病毒EV,并进行EVA71、CVA6、CVA16病毒分型的引物对,经检测四种引物对不产生相互干扰;将PCR体系率先配置成混合体系,在使用时无需单独添加,而只需要加入样本,简化操作过程,降低污染风险;
2、一种肠道病毒假病毒的制备方法,经该制备方法得到的EVA71、CVA6、CVA16假病毒,无复制活性,生物安全性高,能够为手足口病的研究和疫苗评价提供强有力的筛选工具,具备广泛的应用价值。
附图说明
图1干扰试验中的混合样本扩增曲线图;
图2为Western blotting检测目的基因克隆慢病毒载体验证图;
图3为假病毒核酸、假病毒原液扩增曲线图。
具体实施方式
以下的实施例便于更好地理解本发明,但并不限定本发明。下述实施例中的实验方法,如无特殊说明,均为常规方法。下述实施例中所用的试验材料,如无特殊说明,均为自常规生化试剂商店购买得到的。所用引物和探针序列合成由上海生工生物工程有限公司完成;质粒对照品由安徽通用生物完成。
下面结合实施例对本发明的特征和性能作进一步的详细描述。
实施例1
(一)肠道病毒通用和分型RT-PCR检测试剂盒的制备
按下表1配制Q-PCR反应液:
表1 Q-PCR反应液
配制RT-PCR酶:由酶系Hotstart HiTaq&Script III和4×RT PCR Buffer组成。1人份体系中酶系Hotstart HiTaq&Script III 1.2μL,4×RT PCR Buffer 7.5μL。故50人份RT-PCR酶需酶系60μL和Buffer 375μL混合435μL。
配制阳性对照:将含有肠道病毒EVA71、CVA6、CVA16型和EV的特异性基因片段的质粒稀释至10ng/μl,按1:1:1:1混合稀释至1ng/μl,再按10倍比稀释至100pg/μl分装于1.5ml螺旋管中,每管200μl。
分装阴性对照:将灭菌的DEPC水,于1.5ml螺旋管中,每管200μl。
DEPC水:分装DEPC水,于1.5ml螺旋管中,每管500μl。
本发明肠道病毒通用和分型RT-PCR检测试剂盒包括以下组分:
表2试剂盒组分
(二)干扰试验
样本:由肠道病毒EVA71、CVA6、CVA16型和EV的阳性质粒,分别稀释成106copies/μl浓度。阳性质粒按1:1:1:1混合均匀制备为混合样本。
加样:将制备的EVA71、CVA6、CVA16型和EV的质粒、阴性对照、混合样本分别作为模板加入试剂盒反应体系中4ul,每个样重复3次。
上机:实时荧光PCR仪,按上述表2的反应程序上机,实验结果Ct值如表3所示。
表3扩增Ct值
参考图1,根据多重和单重引物的实验扩增结果显示,EVA71、CVA6、CVA16分型和EV的引物之间未造成互相干扰,样本间也未发生干扰影响结果。
实施例2
(一)肠道病毒假病毒的制备方法
Western blot验证质粒表达:
(1)准备293T细胞:用含体积分数10%胎牛血清的DMEM培养液,37℃5%CO2培养箱培养293T细胞,至培养板的细胞80%时进行转染;
(2)脂质体稀释:取120μL Opti-MEM、2ul转染试剂lipo8000和1ug目的质粒,混匀震荡离心5min,室温下静置5min(质粒:转染试剂=1:2=1ug:2ul);
(3)取出12孔板培养的细胞,弃去旧培养基加入新鲜培养基,再将转染复合物加入培养板中轻摇细胞板混合均匀,在37℃,5%CO2培养箱中继续培养48h左右进行细胞转染;
(4)收样:收集细胞并将其裂解,提取蛋白样本。
用Western blot鉴定目的基因质粒在293T细胞中是否表达Flag蛋白,结果如图2所示,表明目的基因质粒构建合适。
病毒包装和收集:
(1)准备293T细胞:用含体积分数10%胎牛血清的DMEM培养液,37℃5%CO2培养箱培养293T细胞,至T25cm2细胞瓶中细胞80%时进行转染;
(2)取转染试剂Lipo8000 12μL,加到300μL Opti-MEM培养基的1.5mL离心管中,混匀后置室温5min。
(3)取pMD2.G、pCMV-dR8.74、目的质粒×flag按6μg:1μg pMD2.G:2μg的pCMV-dR8.74:3μg目的质粒质量比,在另一个1.5mL离心管中混匀质粒;
表4假病毒构建质粒浓度与上样体积
名称 | 浓度ng/μl | 质粒体积 |
CVA-16(VP1) | 635.5 | 4.72μL |
CVA-6(VP1) | 674.0 | 4.45μL |
EVA-71(VP1) | 641.5 | 4.68μL |
pMD2.G | 927.0 | 1.08μL |
pCMV-dR8.74 | 682.5 | 2.93μL |
(4)将混匀的质粒加到转染试剂的管中,混匀后在室温静置5min;
(5)将质粒和转染试剂混合液加到T25cm2细胞瓶中,置于37℃、5%CO2培养箱中培养48h左右,反复冻融2次,收集毒液。
(二)假病毒的验证
分别以上述制备的假病毒原液和假病毒中提取的RNA为模板,使用实施例1的试剂盒进行扩增,具体步骤如下:
(1)用病毒核酸提取试剂盒提取制备假病毒的RNA;
(2)按照本发明试剂盒说明书,计算待测样本的数量N,混合对应的N+2的反应试剂,分装至8连排管;
(3)加样模式:4μl模板(假病毒原液、假病毒核酸、阳性对照、阴性对照),加入含有PCR反应液的8连排管中。
反应程序:按下表5设置反应,在实时荧光PCR仪中上机。等待反应结束。根据实时荧光曲线图判定结果。
表5反应程序
结果判断:阳性检测结果Ct≤35,且有明显指数增长期;阴性检测结果>35,无指数扩增期。
表6扩增Ct值
由图3的结果显示,未提取的假病毒原液扩增结果为阴性,假病毒EVA71、CVA6、CVA16提取的RNA荧光PCR扩增结果为阳性,说明假病毒中无目的基因质粒残留。
本发明所使用的引物探针及EVA71、CVA6、CVA16和EV特异性基因序列和引物探针序列如表7所示:
表7特异性基因序列和引物探针序列
以上所述实施例仅表达了本申请的具体实施方式,其描述较为具体和详细,但并不能因此而理解为对本申请保护范围的限制。应当指出的是,对于本领域的普通技术人员来说,在不脱离本申请技术方案构思的前提下,还可以做出若干变形和改进,这些都属于本申请的保护范围。
Claims (8)
1.一种肠道病毒通用和分型RT-PCR检测试剂盒,其特征在于,包括通用EV、EVA71、CVA6、CVA16的特异性引物、探针组混合液;酶混合液;阳性对照品;阴性对照品组成。
2.根据权利要求1所述的一种肠道病毒通用和分型RT-PCR检测试剂盒,其特征在于,所述EVA71的特异性引物、探针组包括如SEQ ID NO:2~SEQ ID NO:4所示的引物1、2和探针1;
所述CVA6的特异性引物、探针组包括如SEQ ID NO:6~SEQ ID NO:8所示的引物3、4和探针2;
所述CVA16的特异性引物、探针组包括如SEQ ID NO:10~SEQ ID NO:12所示的引物5、6和探针3;
所述通用EV的特异性引物、探针组包括如SEQ ID NO:14~SEQ ID NO:16所示的引物7、8和探针4。
3.根据权利要求1所述的肠道病毒通用和分型RT-PCR检测试剂盒,其特征在于,所述探针1标记FAM荧光,所述探针2标记HEX荧光,所述探针3标记ROX荧光,所述探针4标记CY5.5荧光。
4.一种肠道病毒假病毒的制备方法,其特征在于,所述的肠道病毒EVA71、柯萨奇病毒CVA6、CVA16的VP1基因分别连入慢病毒载体,转染293T细胞,收集假病毒颗粒,纯化假病毒颗粒。
5.根据权利要求4所述的假病毒的制备方法,其特征在于,所述的编码EVA71衣壳蛋白的VP1基因核苷酸序列如SEQ ID No.1所示;
CVA6衣壳蛋白的VP1基因核苷酸序列如SEQ ID No.5所示;
CVA16衣壳蛋白的VP1基因核苷酸序列如SEQ ID No.9所示。
6.根据权利要求4或5所述的假病毒的制备方法,其特征在于,包括以下步骤:
(1)将GeneBank中已经的收录的VP1基因完整翻译蛋白片段5’端、3’端添加相应酶切位点和Flag标签,与慢病毒载体pLVX-Puro连接合成目的质粒;
(2)将步骤(1)所述的目的基因质粒转染到293T细胞48h后,收集细胞并将其裂解,提取蛋白样本,Westernblot验证质粒表达;
(3)将所述目的质粒、包装质粒pCMV-dR8.74、包膜质粒pMD2.G混合、转染293T细胞后获得重组假病毒。
(4)将步骤(3)所述假病毒利用PEG进行浓缩纯化,制备假病毒。
7.根据权利要求6所述的假病毒的制备方法,其特征在于,所述步骤(3)的转染时所需质粒量为6ug,即包膜质粒pMD2.G、包装质粒pCMV-dR8.74、目的质粒×flag的质量比1:2:3。
8.根据权利要求6所述的假病毒的制备方法,其特征在于,所述步骤(3)、步骤(4)中,将转染后293T细胞,在含10%FBS的DMEM培养基中在37℃、5%CO2条件下继续培养48h,培养结束后收取上清液,经离心、过滤后得到所述假病毒。
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