CN107663552A - 西尼罗病毒焦磷酸测序方法 - Google Patents
西尼罗病毒焦磷酸测序方法 Download PDFInfo
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Abstract
本发明属于生物技术领域,它确立了西尼罗病毒焦磷酸测序方法,含有三个技术要点:①确立了核酸检测所需要的引物序列;②确立了检测反应种类;③确立了检测反应体系和反应条件。该西尼罗病毒焦磷酸测序方法可以用于西尼罗病毒科学研究,也可以用于动物或人的临床诊断检测。
Description
技术领域
本发明属于动物病原检测技术领域,涉及一种采用焦磷酸测序(Pyrosequencing)技术检测西尼罗病毒的方法,在科学研究、医学临床诊断和兽医临床诊断上有较大的使用价值。
背景技术
西尼罗病毒病是由西尼罗病毒(West Nile Virus,WNV)引起的一种人畜共患的自然疫源性的虫媒传染病,因于1937年首次从非洲乌干达西尼罗河地区一名发热的妇女血液中分离到病原而得名。传播媒介主要是库蚊,宿主包括鸟类、马牛等哺乳动物和人类。该病分布广泛,已在非洲、中东、西亚和中亚、澳大利亚、北美等地发生和流行,引起人、马和鸟类的大量感染和死亡,发病率、死亡率逐年增加,给发生国家造成重大危害,目前,这一病毒已经引起了人们的高度重视,WHO将其列为当前全球的重大流行病之一。在国务院颁布的《国家中长期动物疫病防治规划(2012—2020年)》中,西尼罗河热被确定为未纳入病种目录,但传入风险增加的动物疫病之一。
该病的实验室检测方法有病毒分离、RT-PCR、ELISA抗体检测、免疫组织化学、中和试验等。病毒分离是最可靠的确诊方法,但是耗时长,工作繁琐。RT-PCR方法特异性很高、检测成本低,但敏感性较差,可能出现假阳性结果,需要对扩增产物进行序列测定。ELISA抗体检测容易出现假阳性等等。因此,建立一种快速有效的鉴别西尼罗病毒的检测方法,对于开展疫病的早期诊断、防止疫情进一步扩散蔓延具有重要意义。焦磷酸测序(Pyro sequencing)技术是新一代DNA序列分析技术,该技术无须进行电泳,DNA片段也无须荧光标记,是一种通用型技术平台。该技术具有操作简便、检测成本低、所需样品量小、快捷、准确、高通量等特点,符合临床大样本检测要求。本研究成果尚未公开发表涉及本发明内容的文章。
发明内容
本发明所要解决的技术问题是提供一种西尼罗病毒E基因的焦磷酸测序技术检测确诊方法,以克服现有检测技术的不足。
为解决上述技术问题,本发明的主要原理是利用焦磷酸测序技术,通过测序引物和聚合酶链式反应扩增单链脱氧核糖核酸模板杂交,与脱氧核糖核酸聚合酶、腺嘌呤核苷三磷酸(ATP)硫酸化酶、荧光素酶、三磷酸腺苷双磷酸酶和底物(APS)、荧光素孵育,逐一加入四种三磷酸碱基脱氧核苷酸(dNTPs):三磷酸腺嘌呤脱氧核苷酸dATP、三磷酸胸腺嘧啶脱氧核苷酸dTTP、三磷酸胞嘧啶脱氧核苷酸dCTP、三磷酸鸟嘌呤脱氧核苷酸dGTP,如与模板配对,此三磷酸碱基脱氧核苷酸与引物的末端形成共价键,释放焦磷酸基团(PPi);腺嘌呤核苷三磷酸(ATP)硫酸化酶在底物(APS)存在的情况下催化焦磷酸形成腺嘌呤核苷三磷酸,腺嘌呤核苷三磷酸驱动荧光素酶介导的荧光素向氧化荧光素转化,氧化荧光素发出与腺嘌呤核苷三磷酸量成正比的可见光信号;光信号由电荷耦合器件(CCD)收集并由软件转化为峰;每个光信号的峰高与反应中掺入的核苷酸数目成正比,腺嘌呤核苷三磷酸和未掺入的三磷酸碱基脱氧核苷酸由三磷酸腺苷双磷酸酶降解,淬灭光信号,并再生反应体系,利用光信号转化得到的峰图,对样品中的目标基因进行准确的检测。
附图说明:
图1是PCR扩增引物特异性实验结果电泳图,M:DNA marker ;1:RT- PCR扩增结果。图2是焦磷酸测序结果图。
具体实施方式
下面通过实施例,说明本发明的技术方案,但本发明的保护范围不限于这个实施例。
本实施焦磷酸测序技术检测西尼罗病毒的方法,包括下列步骤:
首先依据西尼罗病毒E基因为模板,在特定位点进行定点突变以区分野毒株,以防污染导致假阳性。由生物公司合成上述cRNA模板序列并克隆至PCII,获得重组质粒PCRII-WNV-E。以质粒PCRII-WNV-E为模板,利用引物W37-WNVEFR:TTT TTT TTT TTT TTT AGC GTG CACGTT CAC GGA和W41-WNVEFF:TAA TAC GAC TCA CTA TAG GGC GA ACC CTAT GGC GTC TGTTCA进行PCR扩增,得到长度约600bp的带T7启动子序列的产物片段。切胶回收,定量。按体外转录试剂盒Transcript AidTM T7High Yield Transcription Kit(Fermentas#k0441)的说明进行体外转录。-80℃冻存,用作标准品。然后以西尼罗病毒E基因的特异性引物进行反转录聚合酶链式反应(RT-PCR)扩增;用琼脂糖凝胶电泳对扩增产物进行检测,若引物扩增片段长度为183bp,则制备焦磷酸测序单链模板,再进行焦磷酸测序反应,最后根据RT-PCR扩增结果和焦磷酸测序结果来判定样品中是否含有西尼罗病毒。
设计的焦磷酸测序技术检测西尼罗病毒的引物:
上游引物(W17-WNVJF):5'-AAG TTT CTT GGG ACT CCC-3';
下游引物(W20-WNVJR-BIO):5'-CTT AGC GTT GGC CGT GGC-3',5’端生物素标记
测序引物(W22-WNVJFseq):CAG ACA CAG GTC ACG。
具体包括下列步骤:
1体外转录模板的制备
以合成的E基因为模板,用含有T7启动子的扩增引物W37-WNVEFR和W41-WNVEFF扩增WNVE基因保守区。反应体系为:模板1μl,PCR Mix 25μl,上下游引物各1μl,最后加dd H 2O至50μl。反应条件为:94℃预变性2min,94℃变性30s,56℃退火30s,72℃延伸1min30s,共35个循环,72℃延伸10min进行扩增,预期扩增片段长度为600bp。PCR结束后,进行1.5%琼脂糖凝胶电泳凝胶成像观察结果。切下阳性条带,用胶回收试剂盒回收目的片段,用核酸浓度测定仪定量。-20℃保存备用。
2体外转录及纯化
用购自Fermentas生物试剂公司TranscriptAid T7High Yield Transcription kit进行体外转录:
1)转录
5×Transcript AidTM Realtion Buffer 4μL、ATP/CTP/GTP/UTP Mix 8μL、Contro/模板(DNA 1μg、TranscriptAidTMEnzym Mix 2μL,加DEPC-treated water至20μL。置于37℃2-3h,2)纯化前处理去除DNA模板:加2μL DNaseI RNase freed 37℃15min去除DNA,加2μL0.5M EDTA(pH8.0)65℃灭活10min。
2)纯化
(1)加入加115ul DEPC处理水和15ul 3M NaAC,混匀涡旋。
(2)加等体积的酚:氯仿(1:1)(取下层)混匀,离心12000rpm,10min;
(3)吸取上清,再加等体积的氯仿,混匀,离心12000rpm,10min;
(4)吸取上清,加2倍体积的无水乙醇,颠倒混匀放-20℃至少30min,沉淀RNA;
(5)离心12000rpm,10min,弃掉上清;
(6)加70%的冷乙醇500ul洗一次,离心12000rpm,10min,弃掉上清;
(7)40ul DEPC水溶解悬浮RNA(-20℃或-70℃保存);
(8)电泳分析及测浓度
10倍稀释(1ul RNA+9ul DEPC处理水)混匀取4ul样品,加4ul 2×RNA Loading DyeSolution,混匀70℃10min,再冰浴3min,1.5%的胶电泳。10倍稀释剩余的测浓度。计算RNA浓度,配成RNA标准品。
3RT-PCR扩增
将以上体外转录模板稀释108倍,进行RT-PCR扩增,RT-PCR反应体系(50μL):体外转录稀释的模板2ul、2×One Step buffer 25ul、W17-WNVJF1μL、W20-WNVJR-BIO 1μL、E Mix 2μL、RNase free H2O 19μL总体系50μL,50℃30min,94℃预变性2min,94℃变性30s,58℃退火30s,72℃延伸30s,扩增50个循环;最后72℃补充延伸7min。扩增反应后取5μLRT-PCR产物用1.5%的琼脂糖凝胶电泳,凝胶成像观察结果。PCR产物经凝胶成像观察无杂带,大小正确约183bp,可用于测序。RT-PCR产物胶回收后进行浓度测定。
4焦磷酸单链模板制备
将PCR产物50μL转移至加有Sepharose beads 3ul和Binding Buffer 47ul的96孔PCR板中,常温1300rpm-1400rpm混匀10min,让Sepharose beads与PCR产物充分结合;打开真空泵,依次将Vacuum Prep Tool在超纯水中清洗30S、在96孔PCR板中抓取Sepharose beads,随后分别在70%乙醇、Denaturation Buffer、Washing Buffer中清洗10~15s,再将其放人预先加入45μL含0.6μM测序引物的Annealing Buffer的PSQ96孔板中,充分震荡摇动释放Sepharose beads。将Vacuum prep tool移到上方对准含有测序引物的PSQ96孔板,关掉抽真空开关,再将Vacuum prep tool放入含有测序引物的PSQ96孔板摇动,释放结合到Sepharose beads上的单链PCR产物至PSQ96板中。
5焦磷酸测序反应
将放有样品的PSQ96孔板80℃孵育2min,冷却至室温后进行焦磷酸测序反应。根据指示图及提示将酶混合物、底物混合物和四类核苷酸(A、T、C和G)分别加入试剂舱固定位置。设定程序,将试剂舱和PSQ96孔板放入PSQ TM 96 MD Sys tem仪器中进行测序反应。根据PSQTM 96MA System仪器的软件说明在试剂舱中分别加入的底物APS、dNTP和酶混合物(DNA聚合酶、荧光素酶、ATP硫酸化酶和三磷酸腺苷双磷酸酶);然后将试剂舱和PSQ 96板放入,进行Pyrosequencing反应。据指示图及提示分别加入A、T、G、C、E、S,E、S加入量407/3=136μL/孔,A、T、G、C加入量413/3=138μL/孔,程序顺序:C、A、T、G。
敏感性试验
以体外转录产物为模板,用优化好的RT-PCR反应条件进行扩增,取5μL扩增产物进行琼脂糖凝胶电泳鉴定,并进行浓度测定。将RT-PCR产物进行2倍、4倍、8倍、16倍、32倍、64倍、128倍、256倍稀释后,进行焦磷酸测序反应,确定试验的敏感性。鉴定结果与预期相符,说明所用扩增引物和测序引物敏感性很好。
重复性试验
将扩增的RT-PCR产物分别进行3次焦磷酸测序,比较每次测得的序列结果,确定试验的重复性和稳定性。
Claims (8)
1.西尼罗病毒焦磷酸测序方法,其特征在于先进行引物设计,设计西尼罗病毒的特异性引物及焦磷酸测序引物,再制备RNA标准品,用特异性引物进行逆转录-聚合酶链式反应(RT-PCR),通过琼脂糖凝胶电泳检测扩增产物,若特异性引物扩增片段长度为183bp,则制备焦磷酸测序单链模板,进行焦磷酸测序反应,最后根据RT-PCR扩增结果和焦磷酸测序结果判定样品中是否含有西尼罗病毒。
2.根据权利要求1所述的西尼罗病毒焦磷酸测序方法,其特征在于所述的引物设计:选取西尼罗病毒E基因序列,根据MEGA软件的核苷酸序列比对结果,设计上游引物、下游引物和测序引物,上游引物、下游引物扩增片段长度为183bp,引物序列分别为:
上游引物(W17-WNVJF):5'-AAG TTT CTT GGG ACT CCC-3',5’进行生物素标记;
下游引物(W20-WNVJR-BIO):5'-CTT AGC GTT GGC CGT GGC-3'
测序引物(W22-WNVJFseq):CAG ACA CAG GTC ACG。
3.根据权利要求1所述的西尼罗病毒焦磷酸测序方法,其特征在于所述的提取待测样品RNA是使用商业化RNA提取试剂盒。
4.根据权利要求1所述的西尼罗病毒焦磷酸测序方法,其特征在于所述的用特异性引物进行一步法RT-PCR反应:体外转录稀释的模板2ul、2×One Step buffer 25ul、W17-WNVJF 1μL、W20-WNVJR-BIO1μL、E Mix 2μL、RNase free H2O 19μL总体系50μL;反应程序50℃反转录30min,94℃预变性2min,进入94℃变性30s,58℃退火30s,72℃延伸30s,扩增50个循环;最后72℃补充延伸7min。
5.根据权利要求1所述的西尼罗病毒焦磷酸测序方法,其特征在于所述的RT-PCR扩增产物电泳检测:取1.5g琼脂糖于100ml电泳缓冲液中加热,充分熔化,加入核酸染料GoldView至终浓度为0.5μg/ml,制成1.5%的琼脂糖凝胶胶;将5μL RT-PCR产物与适量加样缓冲液混合,点样,5V/cm恒压电泳,直至溴酚蓝指示剂迁移至凝胶中部;凝胶成像仪观察电泳结果。PCR产物经凝胶成像观察无杂带,大小正确约183bp,可用于后期测序检测。RT-PCR产物胶回收后进行浓度测定。
6.根据权利要求1所述的西尼罗病毒焦磷酸测序方法,其特征在于所述的制备焦磷酸测序单链模板是使用50μL 8倍稀释的带生物素标记的RT-PCR回收产物、Sepharose beads3ul和Binding Buffer 47ul混合,在室温条件下1400rpm振荡10min;让Sepharose beads与PCR产物充分结合;打开真空泵,依次将Vacuum Prep Tool在超纯水中清洗30S、在96孔PCR板中抓取Sepharose beads,随后分别在70%乙醇、Denaturation Buffer、Washing Buffer中清洗10~15s,再将其放人预先加入45μL含0.6μM测序引物的Annealing Buffer的PSQ96孔板中,充分震荡摇动释放Sepharose beads。将Vacuum prep tool移到上方对准含有测序引物的PSQ96孔板,关掉抽真空开关,再将Vacuum prep tool放入含有测序引物的PSQ96孔板摇动,释放结合到Sepharose beads上的单链PCR产物至PSQ96板中。将样品置于80℃烘箱2min,再冷却至室温。
7.根据权利要求1所述的西尼罗病毒焦磷酸测序方法,其特征在于所述的焦磷酸测序反应是在28℃下于焦磷酸测序仪PyroMark ID上检测,根据指示图及提示将酶混合物、底物混合物和四类核苷酸(A、T、C和G)分别加入试剂舱固定位置。设定程序,将试剂舱和PSQ96孔板放入PSQ TM 96MD Sys tem仪器中进行测序反应。根据PSQTM 96MA System仪器的软件说明在试剂舱中分别加入的底物APS、dNTP和酶混合物(DNA聚合酶、荧光素酶、ATP硫酸化酶和三磷酸腺苷双磷酸酶);然后将试剂舱和PSQ 96板放入,进行Pyrosequencing反应。据指示图及提示分别加入A、T、G、C、E、S,E、S加入量407/3=136μL/孔,A、T、G、C加入量413/3=138μL/孔,程序顺序:C、A、T、G。加样使用600mbar/8msec的加样压力和时间,每轮反应时间65s;引物链随着不同三磷酸碱基脱氧核苷酸(dNTP)的加入而延伸,随着核酸的结合,CCD摄像机检测到发出的光信号。
8.根据权利要求1所述的西尼罗病毒焦磷酸测序方法,其特征在于所述的根据RT-PCR扩增结果和焦磷酸测序结果判定样品中是否含有西尼罗病毒,若RT-PCR产物经电泳检测未见183bp扩增片段,则样品中不含西尼罗病毒,若RT-PCR产物经电泳检测出现183bp扩增片段,则进行焦磷酸测序分析;若测序结果与序列相符则被检样品中含有西尼罗病毒。
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