CN114058739B - 检测猪捷申病毒环介导等温扩增引物组及试剂盒 - Google Patents
检测猪捷申病毒环介导等温扩增引物组及试剂盒 Download PDFInfo
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Abstract
本发明公开了一种检测猪捷申病毒(porcine tescho virus,PTV)环介导等温扩增引物组及试剂盒。本发明根据PTV保守基因组序列设计了1对外引物F3和B3,1对内引物FIP和BIP,所述的引物组中各引物序列如SEQ ID NO.1‑4所示。此外,根据所设计的引物建立了一种PTV的环介导等温扩增(PTV‑LAMP)方法。该PTV‑LAMP检测方法的放大产物可以直接可视化,以颜色变化观察。本发明建立的PTV‑LAMP方法的检测下限为2.86copy/μL,与除猪捷申病毒之外的其它病毒没有交叉反应,特异性强,灵敏性高,无需昂贵仪器设备,便于在资源有限的农村诊所和野外情况下使用。
Description
技术领域
本发明涉及一种检测猪捷申病毒的环介导等温扩增引物组及试剂盒,属于动物传染病学检测技术领域。
背景技术
猪捷申病毒(porcine tescho virus,PTV)属于小RNA病毒科的捷申病毒属,会引起一种接触性的病毒性传染病,即猪捷申病(Teschen disease)。该病毒会导致猪出现脑脊髓灰质炎、母猪繁殖障碍、腹泻、心包炎、心肌炎、皮肤损伤和肺炎等症状。猪捷申病分布的地理区域比较广泛,目前,在世界各地都有报道猪捷申病,猪捷申病已经成为世界范围的猪传染病之一,影响世界养猪业的正常生产,猪捷申病的调查、诊断、预防和疫苗研发等相关研究值得广大研究者关注。
已发现的家猪PTV血清型分别为PTV-1~PTV-12共12个血清型,在野猪中检出PTV-13,故目前共有13种血清型毒株。不同血清型毒株在基因序列存在差异,13个血清型毒株可形成各自的进化树分支。据报道,不同血清毒株的毒力也存在差异,导致的临床症状也不同。PTV-1的强毒株可导致严重的捷申病毒脑脊髓灰质炎,被世界动物卫生组织(OIE)列为B类传染病,PTV-1的其他弱毒株及其他血清型能引起温和型或隐性感染。
猪是PTV的唯一宿主,不同品种不同日龄猪只均可感染,其中血清抗体阴性猪较为易感,幼龄猪的易感性最高。PTV病具有地方流行性,隐性感染猪、病猪和康复猪为主要传染源,该病毒在猪群的感染呈波浪式。带毒猪通过排泄物持续散毒,污染饮水和饲料导致其它各个年龄段猪群经消化道、呼吸道等途径感染。未妊娠母猪感染后,带毒期可达2个月,妊娠母猪带毒期约为3个月,可通过胎盘感染胎儿。公猪精液可带毒,但无证据表明精液中携带病毒可感染母猪。
对PTV的流行病学调查研究说明PTV在我国分布广泛,但分布具有地域差异性,各个地区流行的血清型不同。虽然PTV未对我国养猪业造成严重影响,相关研究学者以及研究报道较少,但应对其潜伏感染引起高度重视,一旦PTV出现爆发性流行,将对我国养猪业造成严重损失。
环介导的等温扩增(LAMP)是一种较新的核酸扩增方法,能在等温条件下扩增DNA,具有快速、高效、特异性强等特点。这种方法依赖于BstDNA聚合酶大片段进行的自动循环链置换DNA合成。LAMP方法最大的优点是能够在60℃-65℃的恒温条件下,在1h或更短的时间内扩增出特异性和灵敏度高的特异性靶DNA序列,而且不需要热循环仪或专门的实验室设施。本方法的较高扩增效率使得使用插入染料(如紫外光照射的SYBR Green I或琼脂糖凝胶电泳后的数字成像)可以简单地观察到扩增。因此,LAMP方法已被开发用于检测多种病毒。
发明内容
本发明目的在于提供一种检测PTV的环介导等温扩增引物组及其试剂盒。本发明的提出为检测猪内脏组织和粪样的PTV提供了一种实用而经济的替代方法,甚至可以在野外使用。
本发明目的是通过以下技术方案来实现的:
本发明的一种检测猪捷申病毒(porcine tescho virus,PTV)的环介导等温扩增引物组,所述的引物组由1对外引物F3和B3和1对内引物FIP和BIP组成,所述的引物组中各引物序列如下:
F3:TGGGACTGCATTGCATAT
B3:GGCCCTTATCTTTGCGTT
FIP:GCATTCCCATACAGGAACTCCACCCTAGGCACCTATTGAGA
BIP:GGCTGGCCGTCTGTACTTTGCAGAGAGAAAATTTTCAAACATG。
进一步的,本发明还提出了所述的引物组在制备检测猪捷申病毒的试剂中的用途。
其中,优选的,所述的试剂为用于环介导等温扩增检测的试剂。
再进一步的,本发明还提出了一种检测猪捷申病毒的环介导等温扩增试剂盒,其中含有以上所述的引物组。
其中,优选的,所述试剂盒还包括:荧光目视检测试剂、10×反应缓冲液、BstDNA聚合酶、10mM dNTP mix、betaine、MgSO4、超纯水。
其中,优选的,用于检测猪捷申病毒时,环介导等温扩增的反应体系为25μL,包括:5pmol F3和B3各1μL,40pmol FIP和BIP各1μL,10×反应缓冲液2.5μL,8U/μLBstDNA聚合酶1μL,5.0M betaine 4μL,30mM MgSO45μL,10mM dNTPmix 2.5μL,待检样品cDNA模板2μL,其余用无核酸酶超纯水补足至25μL。
其中,优选的,用于检测猪捷申病毒时,环介导等温扩增的反应条件为63℃反应60min。
本发明还提供了所述的试剂盒在制备检测或诊断猪捷申病毒试剂中的用途。
本发明还提供了所述的环介导等温扩增反应的引物组在制备PTV快速诊断试剂中的用途。
本发明具有以下优点和效果:
1、污染小,结果直观:本发明试剂盒的荧光目视试剂(FD)在反应前加入,污染小。其含有的钙黄绿素起初与锰离子结合而处于荧光粹灭状态。但是随着LAMP反应的进行,被反应副产物中的焦磷酸根离子夺去结合的锰离子,钙黄绿素恢复游离态从而发出荧光,并进而与反应液中的镁离子相结合,使荧光信号得到增强。
2、操作简便、快速:本发明建立的PTV LAMP方法操作简便、无需复杂昂贵仪器。此外,建立的PTV LAMP方法快速高效,从提取样品到结果判定可在60min内完成(时效性和TaqMan实时荧光定量PCR方法相当)。反应结果判定方法简单,LAMP反应的扩增产物用SYBRGreen I染色,在紫外光照射下肉眼可检测到扩增产物。
3、灵敏度高:本发明通过用10倍连续稀释细胞培养中的PTVRNA的方法确定了LAMP的检测限。本发明的PTV LAMP对PTV的检测下限与其他虫媒病毒相似:可检测到2.86copy/μL的PTV RNA。
4、特异性强:本发明的PTV LAMP方法特异性好,用PRV,GETV,PRRSV,PCV,CSFV和JEV的RNA进行PTV LAMP检测,未发现阳性结果。
附图说明
图1为PTV的环介导等温扩增方法建立的琼脂糖凝胶电泳结果;
图2为RT-LAMP检测PTV温度的优化;
其中,1-6泳道:分别对应于65℃、64℃、63℃、62℃、61℃和60℃。M泳道:DNA标记;7泳道:阴性对照;
图3为RT-LAMP检测PTV敏感性的琼脂糖凝胶电泳图;
其中,1-6泳道:PTV RNA浓度从2.86×105到0.286copy/μL;泳道M:DNA marker;泳道7:阴性对照;
图4为RT-LAMP检测PTV的特异性;
其中,M泳道:DNA标记;泳道1-5,LAMP分别来自模板PTV、伪狂犬病毒(PRV),盖塔病毒(GETV),呼吸综合征病毒(PRRSV),猪圆环病毒(PCV),猪瘟病毒(CSFV)和乙型脑炎病毒(JEV)。
具体实施方法
下面进一步描述本发明,本描述中介绍的实施案例仅是范例性的,并不对本发明的范围构成限制。本专业技术人员应该理解的是,在不偏离本发明原理和方法的情况下,对本发明技术方案的细节和形式进行部分修改或替换,但基于此修改或替换均属于本发明的保护范围内。
实施例1
一、实验方法
1试验毒株和菌株
从43份腹泻仔猪体内分离到的猪捷申病毒(PTV)毒株,选择该毒株系作为阳性标准。本实验室分离鉴定了伪狂犬病毒(PRV),盖塔病毒(GETV),猪繁殖与呼吸综合征病毒(PRRSV),猪圆环病毒(PCV),猪瘟病毒(CSFV)和乙型脑炎病毒(JEV)。
2 RT-LAMP引物组的设计与合成
根据GenBank中登录的所有PTV基因序列特征,利用分子生物学软件进行分析比较,选择PTV 5’端的非编码保守区域片段,利用LAMP引物设计的在线网站(https://primerexplorer.jp/lampv5/index.8l)计引物组,包括外引物1对(F3和B3)、内引物1对(FIP和BIP),上述2对引物的序列如下:
F3:TGGGACTGCATTGCATAT(SEQ ID NO:1)
B3:GGCCCTTATCTTTGCGTT(SEQ ID NO:2)
FIP:GCATTCCCATACAGGAACTCCACCCTAGGCACCTATTGAGA(SEQ ID NO:3)
BIP:GGCTGGCCGTCTGTACTTTGCAGAGAGAAAATTTTCAAACATG(SEQ ID NO:4)
将设计好的相关引物,经NCBI数据库进行BLAST分析,符合试验预期。引物由金唯智生物科技有限公司(中国苏州)合成。
利用设计的外引物(F3和B3)(内引物FIP和BIP在外引物扩增区域以内,条带较短,常规PCR检测易和引物二聚体混淆,故本发明仅进行引物BLSAT分析)对PTV进行常规PCR扩增,按照2×TransTaq-T PCR SuperMix说明书进行PCR反应,反应体系参考试剂盒说明书配制,反应体系为50μL,其中2×TransTaq-T PCR SuperMix反应液25μL、上下游引物(F3和B3,10μM)各1μL、提取的RNA反转录成为cDNA加入1μ,补充无核酸酶超纯水22μL至终体积50μL。反应条件为:94℃预变性4min;94℃ 40s,54℃ 30s,72℃ 35s,30个循环;循环结束后72℃延伸10min。反应结束后,进行常规琼脂糖凝胶电泳。结果仅F3和B3对PTV扩增出现特异性目的条带;对其他病原DNA无交叉干扰,说明特异性强。
3 RT-LAMP方法的建立和优化
RT-LAMP反应混合物包含5.0pmol F3和B3引物各1.0μL、40pmol BIP和FIP引物各1.0μL、2.5μL 10×反应缓冲液、1.0μL 8U/μL Bst DNA聚合酶(New England Biolabs)、2.5μL 10mM dNTP混合物(Takara)、4.0μL 5M betaine(Sigma Aldrich)、5.0μL 30mM MgSO4(Thermo Fisher Scientific)、2.0μL靶cDNA和4.0μL无核酸酶超纯水。将混合物分别在60、61、62、63、64和65℃下孵育60min,以确定最佳反应温度。反应在80℃热失活10min后终止。用无菌水代替模板cDNA作为阴性对照。RT-LAMP产物(10μL)经2.5%琼脂糖凝胶电泳分离,溴化乙锭染色紫外可见。重复实验进行三次。
4 RT-LAMP检测方法的特异性试验
使用上述建立的RT-LAMP检测方法对猪捷申病毒(PTV)、伪狂犬病毒(PRV),盖塔病毒(GETV),呼吸综合征病毒(PRRSV),猪圆环病毒(PCV),猪瘟病毒(CSFV)和乙型脑炎病毒(JEV)进行检测。反应在63℃下进行60min,试验重复三次。
5 RT-LAMP检测方法的敏感性试验
用分光光度法测定PTVRNA的量,并其转换为分子copy。以10倍稀释的PTV RNA(2.86×105~2.86×100copy/μL)为模板进行反转录。并将cDNA用作LAMP检测的模板。
6建立的RT-LAMP检测方法的临床应用
检测广东省不同地区的腹泻流产样品43份,在2ML的EP管中加入PBS进行机械研磨,上清液在4℃下12000g离心30min后转移到灭菌的微管中。用上清液进行RT-LAMP检测PTV的阳性率,同时通过病毒分离鉴定和q-PCR进行检测,以此来验证RT-LAMP检测方法的准确性。
RT-LAMP反应体系25μL:5pmol F3和B3各1μL,40pmol FIP和BIP各1μL,10×反应缓冲液2.5μL,8U/μL BstDNA聚合酶1μL,5.0M betaine 4μL,30mM MgSO4 5μL,10mM dNTP mix2.5μL,待检样品cDNA模板2μL,其余用无核酸酶超纯水(4μL)补足至25μL。
二、实验结果
2.1结果判定
RT-LAMP扩增PTV琼脂糖凝胶电泳的DNA产物显示出特征的梯形条带,具有多条带的分辨率,表明最终的RT-LAMP产物是不同茎长的茎环DNA的混合物(图1)。相反,阴性对照缺乏这种特有的多带阶梯模式。
2.2 RT-LAMP检测PTV的最佳反应温度和时间优化
对PTV RT-LAMP检测的最佳反应温度和时间进行了研究。在63℃下的LAMP反应产生了比在其他温度下看到的更强的带强度(图2)。因此,LAMP检测PTV的最佳反应条件为63℃反应60min LAMP试验的特异性检测。
2.3 RT-LAMP检测PTV的灵敏度的检测
PTV RNA的10倍系列稀释度(从105到10-1copy/μL)用于测定灵敏度,阳性反应混合物呈现典型的梯形图案。在三次重复中,100%检测到含有2.86copy/μL RT-LAMP检测方法的特异性试验的样品,而没有检测到更多的稀释样品(假设为0.286copy/μL)(图3)。紫外透光显影显示RT-LAMP的检测下限为2.86copy/μL。
2.4 RT-LAMP检测PTV的特异性的检测
在含有PRV,GETV,PRRSV,PCV,CSFV和JEV的cDNA模板的反应混合物中没有观察到RT-LAMP扩增产物,表明RT-LAMP对PTV(图4)具有高度的特异性。
2.5临床样品的检测
仔猪腹泻样品中PTV病毒的分离鉴定结果和Q-PCR结果与RT-LAMP结果一致,阳性率为2.32%(1/43)。阳性样品的序列分析显示与PTV-2的序列同源性最高,从而证实了RT-LAMP检测的较高灵敏度。
以上所述仅为本发明的较佳实施例,凡依本发明申请专利范围所做的均等变化与修饰,皆应属本发明的涵盖范围。
序列表
<110> 广东方道基因生物科技有限公司
<120> 检测猪捷申病毒环介导等温扩增引物组及试剂盒
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 18
<212> DNA
<213> artificial sequence
<400> 1
tgggactgca ttgcatat 18
<210> 2
<211> 18
<212> DNA
<213> artificial sequence
<400> 2
ggcccttatc tttgcgtt 18
<210> 3
<211> 41
<212> DNA
<213> artificial sequence
<400> 3
gcattcccat acaggaactc caccctaggc acctattgag a 41
<210> 4
<211> 43
<212> DNA
<213> artificial sequence
<400> 4
ggctggccgt ctgtactttg cagagagaaa attttcaaac atg 43
Claims (5)
1.一种检测猪捷申病毒(porcine tescho virus,PTV)的环介导等温扩增引物组,其特征在于:所述的引物组由1对外引物F3和B3和1对内引物FIP和BIP组成,所述的引物组中各引物序列如下:
F3:TGGGACTGCATTGCATAT
B3:GGCCCTTATCTTTGCGTT
FIP:GCATTCCCATACAGGAACTCCACCCTAGGCACCTATTGAGA
BIP:GGCTGGCCGTCTGTACTTTGCAGAGAGAAAATTTTCAAACATG。
2.权利要求1所述的引物组在制备检测猪捷申病毒的试剂中的用途。
3.一种检测猪捷申病毒的环介导等温扩增试剂盒,其特征在于,含有权利要求1所述的引物组。
4.如权利要求3所述的试剂盒,其特征在于,所述试剂盒还包括:荧光目视检测试剂、10×反应缓冲液、BstDNA聚合酶、10 mM dNTP mix、betaine、MgSO4、超纯水。
5.权利要求3或4所述的试剂盒在制备检测猪捷申病毒试剂中的用途。
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