CN116376848A - 一种卤虫病毒及其检测方法和应用 - Google Patents
一种卤虫病毒及其检测方法和应用 Download PDFInfo
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Abstract
本发明提供了一种卤虫类杆状病毒及其检测方法和应用。所述卤虫类杆状病毒(brine shrimp virga‑like virus 1),保藏编号为CCTCC No.V202273。该病毒形态呈杆状,直径约30 nm,长度约300 nm,能够感染卤虫卵及其原代细胞。本发明还提供了上述卤虫类杆状病毒的检测试剂盒,为卤虫及甲壳类水产卤虫类杆状病毒的检测与防控提供新的可能。
Description
技术领域
本发明属于海洋病原微生物检测技术领域,具体涉及一种未分类卤虫病毒及其检测方法和应用。
背景技术
杆状病毒科(Virgaviridae)是一类单股正链RNA病毒,国际病毒分类委员会将杆状病毒科分为7个属,Furovirus,Goravirus,Hordeivirus,Pecluvirus,Pomovirus,Tobamovirus及Tobravirus。发明人首次在全球多地卤虫卵中发现一种分布广泛的未分类病毒,该病毒分类上属于一个全新的病毒,对该病毒暂时命名为卤虫类杆状病毒(brineshrimp virga-like virus 1)。由于卤虫是养殖甲壳动物和鱼类的重要饵料,对此病毒的有效检测是进行该病毒乃至该属其它病毒的追踪、调研、预防和控制的前提,因此,需要提供一种有效的检测病毒的方法。
发明内容
针对目前尚没有针对卤虫类杆状病毒检测方法的问题,本发明提供一种检测卤虫类杆状病毒的方法。
为实现上述目的,本发明采用如下技术方案。
一种卤虫类杆状病毒(brine shrimp virga-like virus 1, BsVV1),属于杆状病毒科(Virgaviridae),保藏编号为CCTCC NO: V202273。所述卤虫类杆状病毒呈杆状,为单股正链RNA病毒,病毒粒子直径约20 nm,长度约300nm。能够感染卤虫卵及其制备的原代细胞。
作为新病毒,国际病毒分类委员会(ICTV)可能会给出其他的命名规则,因此卤虫类杆状病毒(BsVV1)作为该病毒的一个泛称,指代本发明所保护的同名病毒以及所有其他名称所代表的符合基因或多肽序列同源性的病毒。已测定的卤虫类杆状病毒的基因组序列如SEQ ID NO: 1、SEQ ID NO: 56- SEQ ID NO: 65所示。由于BsVV1是RNA病毒,其用于复制自身基因组RNA的依赖于RNA的RNA聚合酶缺少3’-5’外切酶活性,因此该酶在复制过程中没有纠错能力,其基因组的全部或部分存在较大的变异性。该病毒的分类是以依赖于RNA的RNA聚合酶为依据,在不影响该病毒种属分类的前提下,基因组的整体或部分特异性序列的片段内可能存在同源性≥74.5%的变异。
一种检测卤虫类杆状病毒的试剂盒,其检测的核苷酸为:
(a):SEQ ID NO: 1、SEQ ID NO: 56- SEQ ID NO: 65任一所示的RNA;或
(b):(a)转换的DNA;或
(c):(a)和(b)的≥74.5%同源的RNA或DNA;或
(d):与(a)或(b)或(c)或(d)或(e)反向互补的RNA和DNA;或
(e):(a)-(d)任一所示的部分;所述部分的长度为30 nt-3000 nt。
优选的,所述部分的长度为70 nt-1200 nt。
更为优选的,所述部分的序列为:
(e1)SEQ ID NO: 2-3、4-5、6-7、8-9、10-11、12-13、14-15、16-17、18-19、20-21、22-23、24-25、26-27、28-29、30-31、32-33、34-35、36-37、38-39、40-41、42-43、44-45、46-47、48-49、50-51所示的成对引物扩增的序列,或
(e2)成对引物扩增的序列互补序列。
上述试剂盒是以卤虫类杆状病毒的核酸序列为检测目标,采用核酸扩增方法、核酸探针方法进行检测。
进一步的,上述试剂盒还包括1对或1对以上的成对引物。
所述成对引物选自确定碱基的核酸、含简并碱基的核酸或含修饰碱基的核酸,如,包含锁核酸的核酸。为了对≥74.5%同源的核酸序列进行检测,最简单的方式是将存在单核苷酸多样性的突变位点视为相应的碱基的简并位点,从而可用包含多种突变碱基的简并碱基的序列作为引物序列;对于这种简并方式也可用次黄嘌呤或其他等效碱基代替。
所述引物的长度为9 nt-45 nt;优选的,长度为15 nt-30 nt;更优选的,长度为18nt-25 nt。
进一步的,所述引物选自如SEQ ID NO: 2- SEQ ID NO: 51所示的任意成对的核酸序列或其反向互补的核酸序列。所述引物选自SEQ ID NO: 2- SEQ ID NO: 51中任意2条序列之间的核酸序列片段或其反向互补的核酸序列片段。
当包含1对引物时,可采用常规PCR检测、SYBR Green I染色的实时荧光定量PCR检测、数字PCR检测或依赖于解旋酶的等温扩增(HDA)检测。
当包含1对以上引物时,可采用套式PCR或多重PCR检测,套式PCR采用2对嵌套的引物,其中外侧的1对引物作为套式PCR的外引物用于第一步扩增,内侧的1对引物作为套式PCR的内引物用于第二步扩增;多重PCR采用2对或更多对相互独立的引物,对多个位点或不同基因型的卤虫类杆状病毒或其变异体进行检测。
在引物设计中,还可以在引物的5’端包含1 nt-100 nt的非特异性序列,这些非特异性序列可以用于改变引物性能,如改变Tm值或GC含量;也可以是为了将特异性序列固定在载体如基因芯片上的锚定序列;还可以是为了纯化蛋白等的功能性序列,如His标签序列、S-Tag多肽编码序列、Thrombin蛋白酶酶切位点编码序列。
上述试剂盒还可包括核酸探针。
所述核酸探针选自序列如SEQ ID NO: 2- SEQ ID NO: 52所示的核酸或互补的核酸。核酸探针可以用地高辛(DIG)、荧光素或者放射性同位素等进行所有核苷酸或特定核苷酸,如DIG-dUTP,的掺入法标记;也可以用DIG、荧光素、报告基团、荧光淬灭基团或放射性同位素等进行末端修饰标记,例如探针的5’端标记FAM、HEX、VIC等,3’端标记淬灭基团TAMRA等;进行直接对卤虫类杆状病毒或其他变异的同源病毒的基因组核酸进行独立的原位杂交或斑点杂交检测,也可与实时定量PCR技术联合,进行TaqMan探针或Beacon探针的荧光定量PCR检测。
所述的试剂盒的检测方法包括但不限于常规聚合酶链式反应(PCR)、恒温对流PCR、套式PCR、实时荧光PCR、恒温对流实时荧光PCR、数字PCR、斑点杂交、原位杂交、环介导等温扩增(LAMP)、滚环扩增技术(RCA)、单引物等温扩增、依赖解旋酶的等温扩增技术(HDA)、交叉引物扩增技术、核酸快递等温检测放大技术;也可包括但不限于同时对1株或1种以上卤虫类杆状病毒与其同源种或变异种的多重PCR、多重实时荧光PCR、基因芯片、芯片检测;还可以包括但不限于同时对包含卤虫类杆状病毒及其同源种或变异种的多重PCR、多重实时荧光PCR、基因芯片、芯片检测。
一种检测卤虫类杆状病毒的试剂盒,其检测的多肽为:
(A):SEQ ID NO: 1、SEQ ID NO: 56- SEQ ID NO: 65任一的核酸序列翻译的多肽序列及多肽片段;或
(B):与SEQ ID NO: 1、SEQ ID NO: 56-SEQ ID NO: 65任一的核酸序列翻译的多肽序列有≥74.5%同源的多肽序列及多肽片段;或
(C):(A)或(B)的抗原或抗体。
如SEQ ID NO: 1所示基因组序列翻译的氨基酸序列包括:开放阅读框1(ORF1)的起始位点为497,终止位点为12469,共3990个氨基酸;ORF2的起始位点为12554,终止位点为17581,共1675个氨基酸;ORF3的起始位点为17659,终止位点为18186,共175个氨基酸;氨基酸序列分别如SEQ ID NO: 53-55所示。SEQ ID NO: 56-65所示基因组的ORF1-ORF3序列翻译的氨基酸序列分别如SEQID NO: 66-95所示。
优选的,所述检测的多肽为:
(A1):SEQ ID NO: 53-55、SEQ ID NO: 66-95任一所示的多肽;或
(B1):与SEQ ID NO: 53-55、SEQ ID NO: 66-95任一所示有≥77.0%同源的多肽;或
(C1):(a1)或(b1)的抗原或抗体。
所述抗原可以是病毒株整体、裂解成分、衣壳、多肽、基因工程蛋白或多肽。所述抗体可以为多克隆抗体、杂交瘤细胞、单克隆抗体或单链抗体。所述核酸适配体可以为使用一个或多个多肽特异性序列经SELEX技术筛选所得的一条或多条核酸序列的单链RNA、单链DNA或单链锁核酸序列,可以通过核酸扩增、核酸克隆或人工合成制备特异性核酸适配体。所述抗原、抗体或核酸适配体可以按照现有技术的方法进行制备。
所述试剂盒的检测方法,可采用竞争性、间接或夹心型免疫反应也可采用固体支持物或免疫沉淀法。所述卤虫类杆状病毒的抗体或抗原性片段可采用现有技术进行标记,如荧光标记、化学发光标记、放射性标记或酶标记。扩增探针信号可以采用生物素和亲和素的方法,酶标记和介导的免疫试验,如ELISA检验。
本发明的保护范围也包括上述卤虫类杆状病毒的抗血清、多克隆抗体、杂交瘤细胞、单克隆抗体、单链抗体或表达单链抗体的菌株或细胞株、核酸适配体或克隆表达生产核酸适配体的菌株。所述抗血清、多克隆抗体、杂交瘤细胞、单克隆抗体、单链抗体或核酸适配体以含有多肽特异性序列的卤虫类杆状病毒的病毒株,病毒株的裂解成份、病毒株的基因工程蛋白或多肽为免疫原按照现有技术的方法进行制备。
如,所述抗卤虫类杆状病毒的抗体为单克隆抗体,可将浓缩的含多肽特异性序列的卤虫类杆状病毒株或其特定抗原片段,如,ORF1、ORF2、ORF3的抗原蛋白施予动物,如,小鼠,视需要可添加适当的佐剂,如:弗氏完全佐剂以进行初次免疫。经适当时间间隔后,视需要以灭活的病毒(或抗原蛋白)及适当的佐剂施与二次免疫。经适当时间间隔后,采集免疫动物的血清,用以评估适合用以采集脾脏细胞的小鼠。从所述适用的小鼠采集脾脏细胞与骨髓瘤细胞,如:FO细胞株、NS细胞株以PEG进行细胞融合。从融合细胞中筛选出具分泌能力的杂交瘤细胞株后,得到融合细胞系,该融合细胞系可分泌抗卤虫类杆状病毒的单克隆抗体。
本发明具有以下优点:
本发明的卤虫类杆状病毒是一种新的卤虫病毒,可感染卤虫。本发明提供了基于卤虫类杆状病毒基因组序列的所建立的核酸扩增、抗原结合等特异性检测方法及基于上述方法所产生的试剂盒、检测设备和检测分析及其应用,为养殖甲壳动物和鱼类鲜活饵料的检测与防控提供新的可能。
生物保藏信息
卤虫类杆状病毒(brine shrimp virga-like virus 1),于2022年09月07日保藏于中国典型培养物保藏中心(CCTCC),保藏地址为中国武汉,武汉大学保藏中心,保藏编号为CCTCC NO: V202273。
附图说明
图1是卤虫类杆状病毒电镜图片;
图2是卤虫类杆状病毒基因组检测的琼脂糖凝胶电泳图;
图3是不同卤虫类杆状病毒分离株的核酸同源性;
图4是不同卤虫类杆状病毒分离株的氨基酸同源性;
图5是根据RdRp构建的卤虫类杆状病毒的系统发育进化树;
图6是实施例2中检测卤虫类杆状病毒试剂盒的2%琼脂糖凝胶电泳图,M为marker,Pos为阳性样品;
图7是实施例3中检测卤虫类杆状病毒试剂盒的荧光定量RT-qPCR扩增曲线图,Pos为阳性样品,Neg为阴性样品。
具体实施方式
下面结合实施例和附图对本发明做进一步说明,但本发明不受下述实施例的限制。
实施例1 卤虫类杆状病毒的分离与鉴定
从不同采集地来源的卤虫卵中分离发现了多株能够感染卤虫卵的病毒,其电镜下形态均相似,如图1所示,该病毒呈杆状,直径约20 nm,长度约300 nm,根据形态特征初步判定均为杆状病毒科病毒。
将收集的不同采集地来源的阳性样品11份,以TRIzolTM法提取RNA后,利用商品化试剂盒去除宿主核糖体RNA,建库,使用Illumina Hiseq进行测序,获得测序数据后分别组装测序结果,使用Genious(version 11.1.5)软件进行分析,发现11株病毒的全基因组核酸同源性最低为74.5%(图2),全基因组氨基酸同源性最低为77.0%(图3),RdRp氨基酸同源性最低为95.5%(图4)。在此基础上设计引物(表1)对全基因组进行长片段的扩增和验证,然后采用SMARTER RACE cDNA Amplification Kit扩增3’末端和5’末端的以获得全基因组序列,如SEQ ID NO:1、SEQ ID NO:56-65所示。
表1 卤虫类杆状病毒基因组测序引物
根据杆状病毒科病毒RNA依赖的RNA聚合酶(RNA-dependent RNA polymerase,RdRp)的氨基酸序列,利用MAFFT软件进行比对,利用MEGA软件使用NJ法构建进化树(图4)。结果显示,该病毒属于杆状病毒科的新种。已对2018年于天津采集的分离株TJ/18的样本进行了保藏,保藏编号为CCTCC NO: V202273。
实施例2 检测卤虫类杆状病毒的PCR试剂盒
按照表2中的序列合成引物,按照表3中的浓度配制各组分组成检测卤虫类杆状病毒的PCR试剂盒:
表2 检测卤虫类杆状病毒的引物序列
表3 检测卤虫类杆状病毒的PCR试剂盒组成
参照以下方法检测:
取携带BsVV1卤虫卵样品20 mg和带有罗氏沼虾野田村病毒(MrNV)、黄头病毒基因8型(YHV-8)、偷死野田村病毒(CMNV)、不携带BsVV1的样品分别于1.5 mL无RNA酶EP管中,研磨后各以TRIzolTM法提取RNA,调整RNA浓度为500 ng/µL,然后反转录为cDNA,然后利用如表4和5所示的体系进行套式PCR扩增:第一轮扩增程序:94℃变性2min;94℃ 30 s、55℃ 30s、72℃ 55 s,30个循环;72℃延伸10min。第二轮扩增程序为:94℃变性2 min;94℃ 30 s、55℃ 30 s、72℃ 30 s,30个循环;72℃延伸10 min。
表4 第一轮PCR扩增体系
表5 第二轮PCR扩增体系
将PCR产物以2%琼脂糖凝胶进行电泳,结果如图5所示。图5(上)中可以观察到702nt的初次扩增条带。图5(下)中可以观察到303 nt的二次扩增条带。图5中,BsVV1(1)、MrNV(2)、YHV-8(3)、CMNV(4)、不携带BsVV1(6)和水(7)在一轮和二轮中均未出现扩增条带,而携带BsVV1的卤虫卵(5)分别在一轮扩增出702 nt的目的条带,二轮扩增出303 nt目的条带。
实施例3 检测卤虫类杆状病毒的荧光定量RT-PCR检测试剂盒
按照表2中的序列合成引物,按照表3中的浓度配制各组分组成检测卤虫类杆状病毒的PCR试剂盒:
按照表6中的序列合成引物和探针,按照表7中的浓度配制各组分组成检测卤虫类杆状病毒1的荧光定量RT-PCR检测试剂盒:
表6 检测卤虫类杆状病毒的荧光定量RT-PCR引物序列及探针序列
表7 检测卤虫类杆状病毒的荧光定量RT-PCR试剂盒组成
参照以下方法进行检测:
按照实施例2中的方法从卤虫类杆状病毒(BsVV1)阳性及阴性的卤虫样品中,以及携带罗氏沼虾野田村病毒(MrNV)、黄头病毒基因8型(YHV-8)、偷死野田村病毒(CMNV)的虾类样品中提取RNA,调整RNA模板浓度为500 ng/µL,然后进行荧光定量RT-PCR扩增。
表8 检测卤虫类杆状病毒的荧光定量RT-PCR反应体系
具体的反应体系与条件如下:
根据样品数量计算出反应体系各成份的体积,在避光条件下加入到1.5mL无RNA酶离心管中,每个样品做3个平行,充分颠倒混匀后快速分装到八连排管中。按顺序将样品RNA加入八连排管,离心后进行定量PCR。具体反应条件如下:55℃反转录10min,95℃变性1min;然后95℃ 10s,60℃ 30s,40个循环。
结果如图6所示:BsVV1和阴性对照品不起峰,阳性对照样品和阳性检测样品起峰。
实施例4 检测卤虫类杆状病毒抗原的ELISA检测试剂盒
1. 试剂盒组成
10:1兔抗卤虫类杆状病毒多克隆抗体包被的96孔板1块、牛血清白蛋白封闭液10mL、160:1小鼠抗卤虫类杆状病毒单克隆抗体0.5 mL、160:1辣根过氧化物酶羊抗鼠IgM二次抗体酶结合物0.5 mL、TMB显色液、2M 硫酸、组织裂解液5 mL、洗涤液(PBST,NaCl 0.8 g,KH2PO40.02 g,Na2HPO4·12H2O 0.29 g,KCl 0.02 g,Tween20 0.05 mL,叠氮钠0.01 g,加双蒸水至100 mL,pH 7.4)100 mL、PBS样品稀释液5 mL、阳性对照1支和阴性对照1支。
2. 检测步骤
(1)包被与封闭:将抗体稀释到适当浓度,每孔加入100 µL,置37℃,4 h;弃去孔中液体(为避免蒸发,板上应加盖或将板平放在底部有湿纱布的金属湿盒中);5%小牛血清置37℃封闭40 min。封闭时将封闭液加满各反应孔,并去除各孔中的气泡,封闭结束后用洗涤液满孔洗涤3遍,每遍3 min。洗涤方法:吸干孔内反应液,将洗涤液注满板孔,放置2 min略作摇动,吸干孔内液,倾去液体后在吸水纸上拍干;
(2)加入匀浆后的待检测样品:检测时采用1:50-1:400的稀释度,保证样品吸取量>20 µL。将稀释好的样品加入酶标反应孔中,每样品至少加双孔,每孔100 µL,置于37℃,孵育60min。用洗涤液满孔洗涤3遍,每遍3min;
(3)加入酶标抗体:根据酶结合物提供商提供的参考工作稀释度进行。37℃,孵育60 min。每孔加100 µL。洗涤同前;
(4)加入底物液:每孔加入TMB-过氧化氢尿素溶液100 µL,置37℃避光放置3-5分钟;
(5)终止反应:每孔加入终止液50 µL终止反应,于20min内测定实验结果;
(6)结果判断:检测450 nm波长下各孔吸光度值,在阳性对照孔平均值≥1.00且阴性对照平均值小于等于0.10的前提下,与CUTOFF值相比较,大于等于0.2的样品判定为阳性,否则判定为阴性。
表9 ELISA检测结果
Claims (10)
1.一种卤虫类杆状病毒,保藏编号为CCTCC NO: V202273。
2.一种检测卤虫类杆状病毒的试剂盒,其特征在于,检测的核苷酸为:
(a):序列如SEQ ID NO: 1、SEQ ID NO: 56- SEQ ID NO: 65任一所示的RNA;或
(b):(a)转换的DNA;或
(c):(a)和(b)的≥74.5%同源的RNA或DNA;或
(d):与(a)或(b)或(c)或(d)或(e)反向互补的RNA和DNA;或
(e):(a)-(d)所示任一核苷酸中长度为30 nt-3000 nt的部分。
3.根据权利要求2所述的试剂盒,其特征在于,所述部分的序列长度为70 nt-1200 nt。
4.根据权利要求2所述的试剂盒,其特征在于,所述部分的序列为:
(e1):SEQ ID NO: 2-3、4-5、6-7、8-9、10-11、12-13、14-15、16-17、18-19、20-21、22-23、24-25、26-27、28-29、30-31、32-33、34-35、36-37、38-39、40-41、42-43、44-45、46-47、48-49、50-51所示的成对引物扩增的序列,或
(e2):(e1)的互补序列。
5.根据权利要求2所述的试剂盒,其特征在于,还包括1对或1对以上的成对引物;所述引物的长度为9 nt-45 nt。
6.根据权利要求5所述的试剂盒,其特征在于,所述引物选自如SEQ ID NO: 2- SEQ IDNO: 51所示的任意2条核酸序列或其反向互补的核酸序列;或,如SEQ ID NO: 2- SEQ IDNO: 51所示的序列以及SEQ ID NO: 2- SEQ ID NO: 51中任意2条序列之间的核酸序列片段或其反向互补的核酸序列片段。
7.根据权利要求5所述的试剂盒,其特征在于,所述引物的5’端连接1 nt-100 nt的非特异性序列;所述非特异性序列用于改变引物Tm值、改变引物GC含量、固定引物于载体上或蛋白分离纯化。
8.根据权利要求5所述的试剂盒,其特征在于,还包括核酸探针;所述核酸探针选自序列如SEQ ID NO: 2- SEQ ID NO: 52所示的核酸或互补的核酸。
9.一种检测卤虫类杆状病毒的试剂盒,其特征在于,检测的氨基酸为:
(A):SEQ ID NO: 1、SEQ ID NO: 56- SEQ ID NO: 65任一的核酸序列翻译的多肽;或
(B):与SEQ ID NO: 1、SEQ ID NO: 56- SEQ ID NO: 65任一的核酸序列翻译的多肽序列有≥74.5%同源的多肽;或
(C):(A)或(B)的抗原或抗体。
10.根据权利要求9所述的试剂盒,其特征在于,所述氨基酸为:
(A1):SEQ ID NO: 53-55、SEQ ID NO: 66-95任一所示的多肽;或
(B1):与SEQ ID NO: 53-55、SEQ ID NO: 66-95任一所示有≥77.0%同源的多肽;或
(C1):(a)或(b)的抗原或抗体。
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Citations (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20040177392A1 (en) * | 2001-09-14 | 2004-09-09 | Ruth Barratt | Crustaceans as production systems for therapeutic proteins |
US20050241011A1 (en) * | 2002-04-09 | 2005-10-27 | Allnut F C T | Enclosed aquacultural systems for production of purified recombinant proteins |
CN101511169A (zh) * | 2005-11-30 | 2009-08-19 | 麻省理工学院 | 病原体检测细胞保存系统 |
CN104080481A (zh) * | 2012-01-31 | 2014-10-01 | 库瑞瓦格有限责任公司 | 包含聚合载体货物复合物和至少一种蛋白或肽抗原的药物组合物 |
CN107810009A (zh) * | 2015-05-15 | 2018-03-16 | 库瑞瓦格股份公司 | 涉及施用至少一种mRNA构建体的初免‑加强方案 |
CN109628638A (zh) * | 2018-12-21 | 2019-04-16 | 中国水产科学研究院黄海水产研究所 | 基于对虾东方病毒基因组序列的检测方法及其应用 |
CN111019909A (zh) * | 2019-12-27 | 2020-04-17 | 中国水产科学研究院黄海水产研究所 | 一种甲壳动物黄病毒及其检测方法和应用 |
CN115322971A (zh) * | 2022-06-14 | 2022-11-11 | 中国水产科学研究院黄海水产研究所 | 一种鱼类圆环病毒及应用 |
-
2022
- 2022-12-13 CN CN202211594129.0A patent/CN116376848A/zh active Pending
Patent Citations (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20040177392A1 (en) * | 2001-09-14 | 2004-09-09 | Ruth Barratt | Crustaceans as production systems for therapeutic proteins |
US20050241011A1 (en) * | 2002-04-09 | 2005-10-27 | Allnut F C T | Enclosed aquacultural systems for production of purified recombinant proteins |
CN101511169A (zh) * | 2005-11-30 | 2009-08-19 | 麻省理工学院 | 病原体检测细胞保存系统 |
CN104080481A (zh) * | 2012-01-31 | 2014-10-01 | 库瑞瓦格有限责任公司 | 包含聚合载体货物复合物和至少一种蛋白或肽抗原的药物组合物 |
CN107810009A (zh) * | 2015-05-15 | 2018-03-16 | 库瑞瓦格股份公司 | 涉及施用至少一种mRNA构建体的初免‑加强方案 |
CN109628638A (zh) * | 2018-12-21 | 2019-04-16 | 中国水产科学研究院黄海水产研究所 | 基于对虾东方病毒基因组序列的检测方法及其应用 |
CN111019909A (zh) * | 2019-12-27 | 2020-04-17 | 中国水产科学研究院黄海水产研究所 | 一种甲壳动物黄病毒及其检测方法和应用 |
CN115322971A (zh) * | 2022-06-14 | 2022-11-11 | 中国水产科学研究院黄海水产研究所 | 一种鱼类圆环病毒及应用 |
Non-Patent Citations (2)
Title |
---|
XUAN DONG等: "Diversity and connectedness of brine shrimp viruses in global hypersaline ecosystems", SCIENCE CHINA LIFE SCIENCES, vol. 67, no. 1, 30 October 2023 (2023-10-30), pages 188 - 203 * |
史成银, 杨冰, 宋晓玲, 徐怀恕: "应用PCR和RT-PCR技术对4种对虾病毒的检测", 海洋水产研究, no. 01, 30 March 2003 (2003-03-30), pages 1 - 5 * |
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