CN113718056A - 用于检测犬粪便中冠状病毒的通用引物及试剂盒 - Google Patents
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Abstract
本发明公开了一种用于检测犬粪便中冠状病毒的通用引物及试剂盒。所述引物序列如下：CCoV‑MF:5’‑GCAACAGGATGGGCTTAC‑3’，CCoV‑7aR:5’‑GCATGGAGGAAAACGAGC‑3’。本发明设计的引物可以增加侦测到犬冠状病毒阳性的比例，也可以减少不必要的测序时间的浪费,对于快速筛检有很大的帮助。
Description
技术领域
本发明属于生物技术领域，具体涉及用于检测犬粪便中冠状病毒的通用引物及试剂盒。
背景技术
CCoV(canine coronavirus)属于α冠状病毒，引起犬传染性肠炎，导致犬轻重不一的腹泻，是对犬危害较大的一种病原体。所有年龄的犬都可感染CCoV，但是幼犬更易感染并且发生临床症状。在一个较大范围的调查中，正常犬CCoV的阳性率达45％，腹泻犬阳性率达61％，在对患胃肠炎的宠物犬的粪便检测中，发现19.5％为CCoV阳性。日本腹泻犬曾分离到8个CCoV毒株，试验证明这些毒株在抗原性方面有很大差异，接种SPF犬发现致病性也不相同。目前CCoV已确定了两个基因型，即CCoV-Ⅰ和CCoV-Ⅱ。一般情况下，CCoV会合并感染犬的其他病原体，造成病情加重，然而几年前报道了一种能够造成犬全身性疾病、致死率很高的变异CCoV-Ⅱ强毒株，即泛嗜性犬冠状病毒(CB/05毒株)。
目前检测犬所使用犬冠状病毒的引物并不多,且目前发表文献所使用的引物在使用上有其缺点:1)DNA片段大小小于500bp,2)容易检测出伪阳性以及3)特异性不佳。有鉴于此,开发用于检测犬冠状病毒的通用引物去检测宠物犬或是流浪犬粪便中冠状病毒是当务之急,并且可以做为之后福建龙岩地区犬冠状病毒流行病学调查的依据。
发明内容
本发明的目的在于提供一种用于检测犬粪便中冠状病毒的通用引物及试剂盒，可以快速筛检宠物犬或流浪犬粪便中冠状病毒的存在并提高筛检阳性率与准确度。
为了实现上述目的，本发明采用的技术方案是：
用于检测犬粪便中冠状病毒的通用引物，所述引物序列如下：
CCoV-MF:5’-GCAACAGGATGGGCTTAC-3’
CCoV-7aR:5’-GCATGGAGGAAAACGAGC-3’
采用上述通用引物检测犬粪便中冠状病毒的方法，包括以下步骤：
1)提取病毒RNA，并反转录获得cDNA；
2)以合成的cDNA为模板，以权利要求1中的CCoV-MF和CCoV-7aR为引物，进行PCR扩增反应，预计扩增的目的片段为1290bp，扩增反应总体积为50uL，PCR反应条件如下：95℃预变性5min，95℃变性1min，56℃退火1min，72℃延伸90s，进行30个循环，最后72℃延伸10min，获得PCR产物；
3)将所得PCR产物置于1％琼脂糖凝胶进行电泳，在紫外投射仪上观察并记录实验结果。
检测犬粪便中冠状病毒的试剂盒，所述试剂盒包括上述通用引物。
本发明的技术思路在于：由于利用PCR技术去检测宠物犬或是流浪犬粪便中是否含有冠状病毒存在伪阳性的缺点，因此还需要搭配1)基因组上不同区域DNA片段的引物，2)全基因组测序再次确认有此病原才能真正证实其病原体的存在。
根据美国国家卫生研究院NCBI网站参考犬冠状病毒M基因以及7a基因序列(登录号:DQ112226.1)设计通用引物以进行PCR：
CCoV-MF:5’-GCAACAGGATGGGCTTAC-3’
CCoV-7aR:5’-GCATGGAGGAAAACGAGC-3’
预计得到1290bp的DNA产物片段。目的条带大于1000bp且横跨3个基因(M,N以及7a基因),可以增加侦测到犬冠状病毒阳性的比例,也可以减少不必要的测序时间的浪费,对于快速筛检有很大的帮助。
申请人已经从福建龙岩新罗区的宠物医院以及流浪动物之家收集250份的粪便检体，利用提取粪便中RNA的试剂进行RNA的抽取，再利用上述引物进行PCR检测犬粪便中冠状病毒的存在，并与已知的全基因组测序的方式去再次验证是否为阳性而非伪阳性，以计算所述引物的准确率
本发明具有以下优点：
1)对引物的GC比例进行优化:PCR反应中可以看出，本发明的引物CCoV-MF有10个GC,CCoV-7aR也有10个GC,GC值相近对于PCR反应的低温退火(Tm)这个步骤，比较容易使引物黏合到模板上,扩增效率也相对提高
2)在引物的尾端黏合进行优化:CCoV-F以及CCoV-7aR这两只引物序列最后以GC最为结尾,GC之间是以三条氢键键结,因此比现有的引物以AT结尾更稳定，在PCR黏合这个步骤相对稳定许多，相对提高PCR扩增的效率
附图说明
图1为采用本发明引物检测犬粪便中冠状病毒的结果。
具体实施方式
1、样品来源:
粪便样品均来自龙岩宠物医院。粪便样品使用50mL离心管4度低温保存当天送回实验室并存在于-80冰箱
2、实验步骤:
2.1样品的前处理
(1)取1g粪便至15ml离心管；
(2)加入9ml无菌PBS，涡旋混匀，-80℃冻存，过夜；
(3)取出，室温/37℃水浴解冻，再置于-80℃冻存3-4h/过夜；
(4)重复步骤3；
(5)取出后室温/37℃解冻，去1ml混悬液至1.5ml离心管中，4℃，1000rpm/min离心10min；
(6)取上清至新的1.5ml离心管，4℃，5000rmp/min离心10min；
(7)取上清用于核酸提取。
2.2样品核酸提取
(1)取300uL前处理上清液中制备的样品上清于无RNA酶的灭菌1.5ml EP管中；
(2)加入1000uL Trizol，充分混匀，室温静置5min；
(3)加入200uL三氯甲烷，充分混匀，室温静置5min，4℃12000rpm离心15min；
(4)离心后，取上清至新的1.5mL EP管中，加入等体积异丙醇，充分混匀，-80℃沉淀隔夜；
(5)4℃12000r离心30min；
(6)小心倒掉上清液,加入600uL,70％的乙醇(DEPC水配置),4℃12000r离心15min,小心倒掉上清液
(7)重复步骤(6)；
(8)用20μl枪头把残余液体吸干；
(9)超净台吹风10分钟风干；
(10)加入50uL无Rnase dH2O溶解沉淀；
(11)跑胶看是否有三个条带(28S,18S,5S rRNA)
(12)将样品保存于-80℃。
2.3 cDNA合成
采用HiScript III 1st Strand cDNA Synthesis Kit试剂盒对提取的病毒RNA进行反转录，获得cDNA。
2.4 PCR扩增
以合成的cDNA为模板,:CCoV-F以及CCoV-7aR当作引物，预计扩增的目的片段为1290bp，扩增反应体系为50μL：含2μL模板，引物正反各1μL,dNTP 2μL,10X Taq buffer 5μL,Taq DNApolymerase(5U/μL)0.5μL，最后补ddH2O 38.5μl)。PCR反应条件如下：第一轮95℃预变性5min，95℃变性1min，56℃退火1min，72℃延伸90s，进行30个循环，最后72℃延伸10min将所得产物置于1％琼脂糖凝胶进行电泳，在紫外投射仪上观察并记录实验结果。
3、实验结果
结果显示可以从这些样本中做出5个目标大小一致的阳性条带(图1)，DNA经过生物公司测序，皆为犬冠状病毒M及N基因。
序列表
<110> 龙岩学院
<120> 用于检测犬粪便中冠状病毒的通用引物及试剂盒
<130> 2021
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 18
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
gcaacaggat gggcttac 18
<210> 2
<211> 18
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
gcatggagga aaacgagc 18
Claims (4)
1.用于检测犬粪便中冠状病毒的通用引物，其特征在于：引物序列如下：
CCoV-MF:5’-GCAACAGGATGGGCTTAC-3’；
CCoV-7aR:5’-GCATGGAGGAAAACGAGC-3’。
2.采用权利要求1所述的通用引物检测犬粪便中冠状病毒的方法，其特征在于：包括以下步骤：
1)提取病毒RNA，并反转录获得cDNA；
2)以合成的cDNA为模板，以权利要求1中的CCoV-MF和CCoV-7aR为引物，进行PCR扩增反应，获得PCR产物
3)将所得PCR产物置于琼脂糖凝胶进行电泳，在紫外投射仪上观察结果。
3.根据权利要求2所述的检测犬粪便中冠状病毒的方法，其特征在于：PCR反应条件如下：95℃预变性5min，95℃变性1min，56℃退火1min，72℃延伸90s，进行30个循环，最后72℃延伸10min。
4.检测犬粪便中冠状病毒的试剂盒，其特征在于：所述试剂盒包括权利要求1所述的通用引物。
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