本发明属于病毒分子检测
技术领域：
，具体涉及一种鲤冠状病毒hl39的数字rt-pcr检测引物及应用。
背景技术：
：套式病毒目(nidovirales)于1996年首次由国际病毒分类委员会(internationalcommitteeontaxonomyofviruses,ictv)提出，该目病毒宿主广泛，可引起哺乳动物、鸟类、爬行动物、鱼虾等水生动物多种重大疾病。套式病毒目冠状病毒科是一类线形单股正链rna病毒，最早于1937年从鸡身上分离出来，almeida等对这些病毒进行了形态学研究，电子显微镜观察发现这些病毒的包膜上有形似日冕的棘突，故提出命名这类病毒为冠状病毒[1]。1975年，国际病毒命名和分类委员会正式命名了冠状病毒科。由冠状病毒科的严重急性呼吸综合征病毒(sars-cov)引起的非典型性肺炎，世界上有30多个国家和地区相继报道出现病例，病死率超过10％。2012年，中东地区开始出现新型冠状病毒，病死率超过50％，引起了世界范围的广泛关注，造成社会对冠状病毒的恐慌。因此，对冠状病毒进行深入研究具有重要意义。2018年ictv调整了套式病毒目的分类，新增了abyssoviridae、medioniviridae、mononiviridae、euroniviridae和tobaniviridae等5个病毒科，其中在tobaniviridae科下新增oncotshavirus属。2017年，申请人发表了鲫冠状病毒的相关研究成果，目前鲤冠状病毒(cyprinidnidovirus,cynv)尚无公开报道，更也无有效的检测方法。申请人首次从鲤鱼病变组织中分离出一株鲤冠状病毒，针对该病毒，设计了数字rt-pcr检测引物，从而实现鲤冠状病毒的微量、快速、精确检测。技术实现要素：本发明的目的在于提供了一种鲤冠状病毒hl39的数字rt-pcr检测引物，所述的引物为f：5'-acctcttacactctgcccct-3'和r：5'-aaagtgccggtggttgagag-3'，所述病毒的保藏编号为cctccno：v201988。本发明的另一个目的在于提供了一种鲤冠状病毒hl39的数字rt-pcr检测引物的应用。所述的引物可用于制备检测鲤冠状病毒hl39的检测试剂盒。为了达到上述目的，本发明采取以下技术措施：一种鲤冠状病毒hl39的数字rt-pcr检测引物，所述的引物为f：5'-acctcttacactctgcccct-3'和r：5'-aaagtgccggtggttgagag-3'；所述病毒的保藏编号为cctccno：v201988。鲤冠状病毒hl39的数字rt-pcr检测引物在制备检测鲤冠状病毒的检测试剂盒中的应用；以上所述的应用中，优选的，所述的鲤冠状病毒hl39的检测试剂盒包括：待测样品的cdna，2×supermix，上下游引物，探针；以上所述的试剂盒中，优选的，所述的探针为：p：5'-fam-agcgcaaaccttcaccggtaaccgccaga-tamra-3'。与现有技术相比，本发明具有以下优点：利用本发明所提供的数字rt-pcr检测方法和试剂盒可以实现对含鲤冠状病毒样品的精准检测，灵敏度可以达到2个拷贝。此外，该方法步骤简便易于操作，因此在实际应用中具有很大的灵活性，是一种检测鲤冠状病毒的有效方法。具体实施方式以下将对本发明的具体实施方式进行详细的说明。应当理解的是，此处所描述的具体实施方式仅用于说明和解释本发明，并不用于限制本发明。本发明所述技术方案，如未特别说明，均为本领域的常规方案，所用试剂或原料，如未特别说明，均购自商业渠道或是已公开。实施例1：一种鲤冠状病毒hl39(本发明或称为cynv-hl39)，其分离过程如下：1)将携带病毒的鲤鱼肝、脑、脾、肾等组织取出并碾磨后，培养基稀释100倍，2000rpm离心后取上清过滤，接种到草鱼性腺细胞(grasscarpovarycellline,gco)单层，25℃培养7天，产生细胞病变效应(cpe)，可见gco细胞收缩变圆、折光度增加，部分细胞开始脱落，细胞单层开始破裂，培养基：m199，10％胎牛血清，ph7.0～7.2。2)收集病变细胞，利用透射电镜进行形态学鉴定。3)收集病毒，对其进行全基因组测序，序列为seqidno.1所示。鲤冠状病毒的形态特征：病毒呈棒状，四周有明显的日冕状棘突结构，与典型的冠状病毒形态相符，该病毒已于2019年11月26日送至中国典型培养物保藏中心进行保藏，分类命名：鲤冠状病毒(cyprinidnidovirus)hl39，保藏编号：cctccno：v201988，地址：中国武汉武汉大学。实施例2：基于鲤冠状病毒hl39的全序列(seqidno.1所示)与其他病毒的全序列的差异设计的数字rt-pcr检测引物，所述的引物如下：f：5'-acctcttacactctgcccct-3'r：5'-aaagtgccggtggttgagag-3'实施例3：利用鲤冠状病毒数字rt-pcr检测引物和探针对鲤冠状病毒的检测，方法包括：1.提取鲤鱼组织或感染细胞总rna并反转录为cdna；2.数字pcr扩增反应1)配制微滴pcr反应体系：微滴pcr扩增反应的反应体系，以20μl计：10μl2×supermix，10μm上下游引物(f和r)各0.8μl，10μm探针0.4μl，cdna模板2μl，ddh2o补足至20μl；所述的探针为：p：5'-fam-agcgcaaaccttcaccggtaaccgccaga-tamra-3'。2)在微滴pcr的生成芯片上，加入步骤1)中的20μl微滴pcr反应体系，再加入70μl的微滴生成用油，进行微滴生成步骤，完成微滴生成步骤后，将微滴化的反应体系转移至96孔板中，封膜。3)将封膜后的96孔板放入pcr扩增仪中，pcr扩增程序为50℃热激活300s；95℃预变性300s；95℃变性15s，60℃退火及延伸60s，共50个循环；完成pcr扩增后，将微滴吸入流式荧光检测器中，在60℃下进行荧光检测。出现明显区别于阴性反应孔的扩增信号，这个反应孔记为阳性扩增孔；阳性样品的判定方法为：在同一组的三个平行组里面，至少出现一个组还有阳性扩增孔，就说明检测样品中含有鲤冠状病毒核酸。阳性质控品反应孔中产生阳性结果以及阴性质控品反应孔中产生阴性结果时，该实验视为有效实验，否则实验无效，需要重新更换反应试剂，并重新进行；当待测样品反应管中为阴性结果时，则说明未检测到鲤冠状病毒核酸；待测样品反应管中为阳性结果，则说明待测样品中含有鲤冠状病毒核酸。实施例4：鲤冠状病毒数字rt-pcr引物特异性实验：利用实施例3所述的方法，对待检病毒样品进行检测，同时设置阴性对照组(双蒸水)和阳性对照组(cynv-hl39)，待检病毒样品具体为：鲤春病毒血症病毒(svcv)，草鱼呼肠孤病毒873(gcrv-873)，草鱼呼肠孤病毒9014(gcrv-9014)，传染性造血器官坏死病毒(ihnv)，病毒性出血性败血症病毒(vhsv)。表1鲤冠状病毒rt-pcr检测试剂盒特异性试验试验编号名称扩增结果1cynv-hl39+2svcv－3gcrv-873－4gcrv-9014－5ihnv－6vhsv－结果显示仅阳性对照组(cynv-hl39)可检测到扩增信号，其余组均无扩增信号，说明鲤冠状病毒数字rt-pcr检测引物具备很强的特异性。实施例5：鲤冠状病毒数字rt-pcr引物灵敏度实验：本实验采用鲤冠状病毒阳性样品cynv-hl39的cdna进行10倍梯度稀释。通过实施例3给出的检测方法进行检测。结果显示本体系在2个拷贝的模板量下，可以产生稳定的扩增，即本数字pcr的检测方法在扩增体系为20ul时，最低可检测到为2个拷贝。虽然，上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述，但在本发明基础上，可以对之作一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。序列表<110>武汉海关技术中心<120>一种鲤冠状病毒hl39的数字rt-pcr检测引物及应用<160>4<170>siposequencelisting1.0<210>1<211>25833<212>dna<213>人工序列(artificialsequence)<400>1ttgaattaccaataaactaccacccgctattatagcactagacactagcgaactgctagc60ttcctccaagctgcacaacgcaaactgtctttttaactctcctaacctcgaacgcagcgc120aggtaccctcacccggaccacgcgtgtgcccttgactggaaaccctaccttcgatcaggc180cttcgaacgcagcaaaggtaccctcacccggaccacttgtgtgcccctacgcctgctctt240acgtagccccatcctacccacccgccaactcttaacctatcagctttgcccaccgtgacg300actagacgtgatctaactgctactaccaatcctattacaaccaacttgcccaccaagacg360actagagagtgacgtaactgctactgcctaaggcactaccagcgaaatcataatctgctc420ctcttgacccaagacggtttggaacggtgctactagtgaactcacttctgctccttgcga480cccaaagacggttattatttggaaaacctcttacactctgcccctttacctactcaatat540aacttaaatctcttttagaatcgcgctagcgcaaaccttcaccggtaaccgccagaacgt600cctacaagctctcaaccaccggcactttgttgctaaatcctacgtgcaagcatctttttg660ctataacatccacgttcgaaaatcaacaaaaatgcccaacatgcgaatccgcaagattgc720tcctaaagccgctgttaaaccaacccatagcggcccaagagtggtctccaatcccgcaag780ctgccacatctacatcggaccccaactgcgtgctcacaactacccgagcgtgctaaaggg840ctcctttaaggctgtcagcgcggaaaccataacagctaccaacctaattgccaccgtccg900caaggatgctggtatttcaggtcaatatgctgggaagatgggttatgaccccgagatggg960acttttcgtctccgtcgaaggtgttcgtgctctccaagcttccaaactccaaactgccct1020taaggcaattaaaaagggtactagaccctttggccttttcaggcctaaacagagtgtgac1080taacacccacgctcaaaatttgctagccctccaccgtgctagctccaccggcccttggac1140caaacacttaccatctcaaggtcccataggtaactgcctcgcagaattcatgcaggagta1200cactaccacaattccaccatttgatagtgtcctcactgaaaacggttggcaatacgctgt1260cacgattgacggtgtgactaaatctcgcctcccgtacaccgaaattcgccgcggtggtta1320tgaatttaaaacaactaattaccacatcggtgcaatccaggttactgccaaccacattgg1380caaagtaaaccctggagatactaaattggactgtcaatatttcatgttggcacgtaatca1440aaagccccaaaacttcactgctgagcaactcgaaggaaacatccataacatcatcttcga1500cctcagtcatgaaactg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