CN114561362A - 一种人源肾综合征出血热汉坦病毒分离方法 - Google Patents
一种人源肾综合征出血热汉坦病毒分离方法 Download PDFInfo
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Abstract
本发明涉及病毒分离技术领域,具体涉及一种人源肾综合征出血热汉坦病毒的分离方法。该方法包括以下步骤:1)Vero‑E6细胞制备;2)样本采集和外周血淋巴细胞制备;3)病毒培养;4)病毒传代;5)病毒鉴定。本发明提出的人源肾综合征出血热汉坦病毒分离方法,采用肾综合征出血热患者外周血淋巴细胞接种Vero‑E6细胞,去除了血液中病毒抗体等成分对病毒分离的影响,能够提高人源汉坦病毒的分离率及分离效率。
Description
技术领域
本发明涉及病毒分离技术领域,具体涉及一种人源肾综合征出血热汉坦病毒的分离方法。
背景技术
肾综合征出血热又称流行性出血热,是由不同种类的汉坦病毒引起的啮齿动物传播疾病,以发热、出血和急性肾功能障碍为主要临床特征。中国占全球报告病例总数的90%以上。病毒分离是汉坦病毒感染诊断的金标准,也是病原学研究的重要技术手段。汉坦病毒分离困难,因其在体外细胞中适应性差,不产生明显的细胞病变效应,分离率低。尤其是人源标本病毒载量相对较低,更难成功分离。
卫生行业标准《流行性出血热诊断标准》(WS278-2008)规定病毒分离采用患者血清样本。常规病毒分离方法采用血清接种Vero-E6细胞,37℃吸附1~2小时去血清,37℃、5%CO2培养21天,阴性盲传3代(共63天),期间3~4天换液一次。但在实际操作中,血清样本分离效率非常低,周期长。
传统的人源汉坦病毒分离率低,因此发明一种分离率高的病毒分离方法对肾综合征出血热的诊断和研究有重要意义。
发明内容
本发明的目的在于克服现有技术中存在的病毒分离率和分离效率低的缺陷,提供一种人源肾综合征出血热汉坦病毒分离方法,能够缩短培养时间,减少换液操作次数,有效提高病毒分离效率和病毒分离率。
为实现上述目的,本发明采用以下技术方案。
一种人源肾综合征出血热汉坦病毒分离方法,包括以下步骤:
1)Vero-E6细胞制备,用细胞生长液将新鲜消化后的Vero-E6细胞稀释为1.0×105~2.0×105个/mL后,加入到T25细胞培养瓶中,每瓶5~7mL,再置于37℃,5% CO2培养箱中培养2~3天,待细胞长至培养瓶底75%~90%,弃培养液,加入细胞维持液,备用;
2)样本采集和外周血淋巴细胞制备,采集肾综合征出血热患者急性期(发病到采样7天以内)抗凝血2~5mL,48h内分离外周血淋巴细胞,具体处理步骤为:抗凝血800g离心10分钟,吸取上清(血浆),加入与上清等体积的PBS缓冲液至血细胞中,充分混匀,获得混合液;在另一个15mL或50mL的离心管里加入与血细胞PBS混合液等体积的淋巴细胞分离液,再缓慢将血细胞PBS混合液加入到淋巴细胞分离液管的上层,500~1000g离心30分钟,离心后液体分为四层,从上到下小心吸取第二层(淋巴细胞层)至一新的离心管,往新的离心管内加入10mL PBS缓冲液,500g离心10分钟,弃上清;继续加入10mL PBS缓冲液将沉淀细胞重悬,500g离心10分钟,离心后弃上清,加入1mL PBS缓冲液重悬细胞,即获得外周血淋巴细胞悬液;
3)病毒培养,将0.5~1.0 mL步骤2)分离得到的外周血淋巴细胞悬液加入至一瓶步骤1)中准备好的Vero-E6细胞中,置于37℃、5% CO2培养箱中培养,每隔7天更换细胞维持液;连续培养至28天为一代;
4)培养物传代,培养物培养至28天后,将瓶内维持液倒掉,加入5mL PBS缓冲液,轻摇细胞培养瓶,弃液体,再吸取0.5mL 0.05% EDTA-胰酶加入至培养瓶中,摇匀,置于37℃,5% CO2培养箱中消化细胞;15~20min后于倒置显微镜下观察,待细胞完全脱落,加入20mL细胞维持液,同时加入3~6mL 1.0×105~2.0×105个/mL新鲜制备的Vero-E6细胞悬液,将细胞吹打混匀后加入到一个新的T75培养瓶中,并置于37℃、5% CO2培养箱中培养;每隔7天更换细胞维持液,连续培养28天;
5)病毒鉴定,采用汉坦病毒荧光RT-PCR法或直接免疫荧光试验进行病毒鉴定;若养物上清进行荧光RT-PCR检测Ct值≤28且随培养时间增加,Ct值出现降低情况,或者采用细胞刮刀刮取少量细胞进行直接免疫荧光试验,出现特异性荧光颗粒,则病毒分离呈阳性,分离培养成功;若培养2代(共56天)结果仍为阴性则可以放弃培养。
优选地,步骤1)中,所述细胞生长液的组分为:88%MEM溶液、5%小牛血清、5% 胎牛血清、1%PS(青霉素1万单位+链霉素10000微克/mL)、1% L-谷氨酰胺(200mM)、用NaHCO3溶液(7.5%)调节pH至7.2~7.4。
优选地,步骤1)中,所述细胞维持液的组分为:96%MEM溶液、1%小牛血清、1% 胎牛血清、1% PS(青霉素1万单位+链霉素10000微克/mL)、1%L-谷氨酰胺(200mM)、用NaHCO3溶液(7.5%)溶液调节pH至7.2~7.4。
优选地,步骤4)中,所述新鲜制备的Vero-E6细胞悬液为胰酶消化后用细胞维持液重悬的Vero-E6细胞,浓度为1.0×105~2.0×105个/mL。
本发明中涉及的化学试剂均为现有产品,均可在市场购得,其中淋巴细胞分离液由天津灏洋华科生物科技有限公司生产销售,MEM、青链霉素、0.05% EDTA-胰酶、L-谷氨酰胺、PBS缓冲液、胎牛血清均由GIBCO公司生产销售,小牛血清购自浙江天杭生物科技股份有限公司。
本发明具有以下有益效果:
(1)本发明提供的人源肾综合征出血热汉坦病毒分离方法,采用的是肾综合征出血热病人急性期外周血淋巴细胞接种敏感细胞进行病毒分离,去除了血液中病毒抗体等成分对病毒分离的影响,能够有效提高病毒分离率,病毒分离率可达40%以上。
(2)本发明所提出的一种人源肾综合征出血热汉坦病毒分离方法,外周血淋巴细胞接种敏感细胞后培养7天后换液一次,28天培养一代,通过胰酶消化自身细胞以及加入新鲜细胞混合传代的方法传代,有利于病毒适应体外细胞,能够提高分离率,最终分离出病毒;仅需培养2代,共56天,缩短了病毒培养时间,提升了病毒分离效率。
(3)本发明所提出的一种人源肾综合征出血热汉坦病毒分离方法,配制的细胞生长液有利于Vero-E6细胞生长,细胞维持液可使Vero-E6细胞在28天内每7天换液一次情况下维持良好状态,同时减少换液操作频次。
附图说明
图1为汉坦病毒荧光PCR结果鉴定结果。
图2为用汉坦病毒荧光素标记抗体对细胞培养物进行直接免疫荧光试验图;
图2中A为阳性分离物的直接免疫荧光试验图,B为阴性对照细胞直接免疫荧光试验图。
具体实施方式
为了能够更清楚地理解本发明的上述目的、特征和优点,下面结合附图和具体实施方式对本发明进行进一步的详细描述。应理解,这些实施例仅用于说明本发明而不用于限制本发明的范围,下列实施例中未提及的具体实验方法,通常按常规实验方法进行。
实施例1 Vero-E6细胞制备
将新鲜消化后的Vero-E6细胞,用细胞生长液稀释成2.0×105个/mL,加入至T25细胞培养瓶中,每瓶添加5mL,然后置于37℃、5% CO2培养箱中培养。培养2~3天待细胞长至培养瓶底75%~80%时,弃培养液,加入细胞维持液,用于后续病毒分离。
实施例2 样本采集和外周血淋巴细胞制备
采集肾综合征出血热患者急性期(即发病到采样7天以内)的抗凝血4ml,24h内分离外周血淋巴细胞。采集的抗凝血800g离心10分钟,吸取上清(即血浆)2mL,加入与上清等体积(即2mL)的PBS缓冲液至血细胞中,充分混匀,获得血细胞PBS混合液;在另一个15mL离心管里加入4ml淋巴细胞分离液;将血细胞PBS混合液加入到淋巴细胞分离液管的上层,800g离心30分钟,离心后液体分为四层,从上到下小心吸取第二层的淋巴细胞层至一新的离心管,往新的离心管内加入10mL PBS缓冲液,500g离心10分钟,弃上清;继续加入10mLPBS缓冲液将沉淀细胞重悬,500g离心10分钟,离心后弃上清,再加入1mL PBS缓冲液重悬细胞,即获得外周血淋巴细胞悬液。
实施例3 病毒培养
将0.5 mL新分离的外周血淋巴细胞悬液加入至一瓶实施例1中准备好的Vero-E6细胞,置于37℃,5%CO2培养箱中培养,每隔7天更换一次细胞维持液。连续培养至28天为一代。
实施例4 培养物传代
上述培养物培养28天(一代)后,将瓶内维持液倒掉,加入5mL PBS缓冲液,轻摇细胞培养瓶,弃液体,再吸取0.5mL的0.05% EDTA-胰酶加入至培养瓶中,摇匀,置于37℃,5%CO2培养箱中消化细胞。15~20min后于倒置显微镜下观察,待细胞完全脱落,加入20mL细胞维持液,同时加入3mL 2.0×105个/mL新鲜制备的Vero-E6细胞悬液,将细胞吹打混匀后加入到一个新的T75培养瓶中,再置于37℃,5% CO2培养箱中培养。每隔7天更换细胞维持液。连续培养28天。
实施例5 病毒鉴定
采用荧光RT-PCR法或直接免疫荧光试验进行病毒鉴定。培养过程中,每次换液前收集培养物上清,进行荧光RT-PCR检测,若养物上清进行荧光RT-PCR检测Ct值≤28且随培养时间增加出现降低情况,或者采用细胞刮刀刮取少量细胞进行直接免疫荧光试验,出现特异性荧光颗粒,则病毒分离阳性。培养2代(共56天)结果仍为阴性则可以放弃培养。
试验例
1 材料和方法
1.1 主要试剂
淋巴细胞分离液(医级)(天津灏洋华科生物科技有限公司),MEM培养基、青链霉素、0.05% EDTA-胰酶、L-谷氨酰胺、PBS缓冲液、胎牛血清(GIBCO),小牛血清(浙江天杭生物科技股份有限公司)。核酸提取试剂(QIAGEN),荧光RT-PCR试剂(Thermo Fisher),荧光素标记的汉坦病毒抗体(第四军医大学)。
1.2 人源汉坦病毒分离
按本发明实施例1~5的步骤分离培养获得。
1.3 荧光RT-PCR鉴定
采用汉坦病毒双重实时荧光PCR方法,引物探针各0.5μL(共3μL),ddH2O 3.5μL,核酸模板5μL。PCR反应条件:45℃10min;95℃ 10min;95℃ 15s,55℃ 45s(采集荧光),45个循环。
1.4 直接免疫荧光试验
用细胞刮刀刮取少量细胞至载玻片,在生物安全柜干燥,至冷丙酮固定20分钟后,用PBS缓冲液冲洗2次,蒸馏水漂洗1次,吹干,加荧光素标记汉坦病毒抗体,置于湿盒内37℃孵育30分钟取出,再用PBS缓冲液冲洗3次,蒸馏水漂洗1次,吹干;甘油封片后在荧光显微镜下观察结果。
2 结果
2.1 人源汉坦病毒分离
按照本发明实施例1~5方法,共对26例HFRS病例急性期抗凝血分离外周血淋巴细胞样本进行汉坦病毒分离,经鉴定,成功分离到12株汉坦病毒,病毒分离率46.15%;而采用常规方法未分离到病毒。
2.2 荧光RT-PCR鉴定
每次换液前收集培养物上清进行核酸提取和荧光RT-PCR鉴定,由图1可见经培养后,培养物上清中核酸Ct值≤28且随培养时间增加出现降低情况。
2.3 直接免疫荧光试验
采用荧光素标记汉坦病毒抗体分别对细胞培养物和未接种病毒的对照细胞培养物进行直接免疫荧光试验,结果表明病毒分离阳性的细胞出现绿色闪亮荧光(见图2中A),对照细胞未见绿色荧光(见图2中B)。
本发明的病毒分离方法采用每隔7天更换一次细胞维持液,连续培养至28天为一代的病毒培养传代方式;7天的周期有利于Vero-E6细胞生长,配制的细胞维持液可使Vero-E6细胞在28天内每7天换液一次情况下良好状态,同时增加了上清中病毒与细胞接触时间,减少换液次数减少了污染的机会,便于有效分离出病毒;病毒体外培养细胞适应能力弱,增加每代的培养周期(28天)可以延长病毒适应时间,提高分离率。综上所述,本发明提出的人源汉坦病毒分离方法,病毒分离率高,病毒培养时间短,能为肾综合征出血热的诊断和研究提供帮助。
Claims (3)
1.一种人源肾综合征出血热汉坦病毒分离方法,其特征在于,包括以下步骤:
1)Vero-E6细胞制备,用细胞生长液将新鲜消化后的Vero-E6细胞稀释为1.0×105~2.0×105个/mL后,加入到T25细胞培养瓶中,每瓶5~7mL,再置于37℃,5% CO2培养箱中培养2~3天,待细胞长至培养瓶底75%~90%,弃培养液,加入细胞维持液,备用;
2)样本采集和外周血淋巴细胞制备,采集肾综合征出血热患者急性期即发病到采样7天以内的抗凝血2~5mL,48h内分离外周血淋巴细胞;分离外周血淋巴细胞制备的具体处理步骤为:将采集的抗凝血800g离心10分钟,吸取上清,加入与上清等体积的PBS缓冲液至血细胞中,充分混匀,获得混合液;在另一个15mL或50mL的离心管里加入与血细胞PBS混合液等体积的淋巴细胞分离液,再缓慢将血细胞PBS混合液加入到淋巴细胞分离液管的上层,500~1000g离心30分钟,离心后液体分为四层,从上到下小心吸取第二层即淋巴细胞层至一新的离心管,往新的离心管内加入10mL PBS缓冲液,500g离心10分钟,弃上清;继续加入10mL PBS缓冲液将沉淀细胞重悬,500g离心10分钟,离心后弃上清,加入1mL PBS缓冲液重悬细胞,即获得外周血淋巴细胞悬液;
3)病毒培养,将0.5~1.0 mL步骤2)分离得到的外周血淋巴细胞悬液加入至一瓶步骤1)中准备好的Vero-E6细胞中,置于37℃,5% CO2培养箱中培养,每隔7天更换细胞维持液;连续培养至28天为一代;
4)培养物传代,培养物培养至28天后,将瓶内维持液倒掉,加入5mL PBS缓冲液,轻摇细胞培养瓶,弃液体,再吸取0.5mL 0.05% EDTA-胰酶加入至培养瓶中,摇匀,置于37℃,5%CO2培养箱中消化细胞;15~20min后于倒置显微镜下观察,待细胞完全脱落,加入20mL细胞维持液,同时加入3~6mL 1.0×105~2.0×105个/mL新鲜制备的Vero-E6细胞悬液,将细胞吹打混匀后加入到一个新的T75培养瓶中,并置于37℃,5% CO2培养箱中培养;每隔7天更换细胞维持液,连续培养28天;
5)病毒鉴定,采用汉坦病毒荧光RT-PCR法或直接免疫荧光试验进行病毒鉴定;若养物上清进行荧光RT-PCR检测Ct值≤28且随培养时间增加,Ct值出现降低情况,或者采用细胞刮刀刮取少量细胞进行直接免疫荧光试验,出现特异性荧光颗粒,则病毒分离呈阳性。
2.根据权利要求1所述的人源肾综合征出血热汉坦病毒分离方法,其特征在于,步骤1)中,所述细胞生长液的组分为:88%MEM溶液、5%小牛血清、5%胎牛血清、1%PS(青霉素1万单位+链霉素10000微克/mL)、1% L-谷氨酰胺(200mM)、用NaHCO3溶液(7.5%)调节pH至7.2~7.4。
3.根据权利要求1所述的人源肾综合征出血热汉坦病毒分离方法,其特征在于,步骤1)中,所述细胞维持液的组分为:96%MEM溶液、1%小牛血清、1% 胎牛血清、1% PS(青霉素1万单位+链霉素10000微克/mL)、1%L-谷氨酰胺(200mM)、用NaHCO3溶液(7.5%)溶液调节pH至7.2~7.4。
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