CN107937609B - 一种检测辣椒脉斑驳病毒专用引物及其检测方法 - Google Patents
一种检测辣椒脉斑驳病毒专用引物及其检测方法 Download PDFInfo
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Abstract
一种检测辣椒脉斑驳病毒专用引物及其检测方法,涉及一种检测病毒引物及其检测方法,检测辣椒脉斑驳病毒所用到的PCR引物具体为:上游引物:ChiVMV‑CP‑F:如SEQ ID NO.1所示,即,5’‑GCACCATACATTTCAGAAACAGC‑3’,下游引物:ChiVMV‑CP‑R:如SEQ ID NO.2所示,即,5’‑GAACCACACTGAAGAATATGRATG‑3’。本发明所提供的PCR引物对ChiVMV的检出具有通用性,不仅能检测出龙葵样品中的ChiVMV,还可以检测到番茄样品中的ChiVMV。在检测过程中利用RT‑PCR手段,具有灵敏性高、操作简便等特点。实验表明,利用本发明检测龙葵和番茄上的辣椒脉斑驳病毒(ChiVMV),有较高的特异性。
Description
技术领域
本发明涉及一种检测病毒引物及其检测方法,特别是涉及一种检测辣椒脉斑驳病毒专用引物及其检测方法。
背景技术
辣椒脉斑驳病毒(Chilli veinal mottle virus, ChiVMV),是Potyvirus的确定种,长约900nm,基因组为单链正义RNA,5’-端具有一个共价结合基因组连接蛋白(viralprotein genome-linked, VPg),3’-端为20-160nt个腺苷酸组成的Poly(A)尾巴。基因组编码一个350 kDa的多聚蛋白(Polyprotein),随后切割成解旋酶、复制酶和衣壳蛋白等8~10个蛋白参与病毒的复制、运动和包装。ChiVMV的传播主要通过虫媒的形式以非持久性进行传播。ChiVMV在亚洲危害严重,给越南、泰国、印度、巴基斯坦、韩国和中国的辣椒产业带来重创。在我国,ChiVMV在海南岛和台湾多地的辣椒和番茄上危害。ChiVMV侵染初期会诱导寄主植物表现为叶脉产生深色条纹、叶肉组织皱缩或叶面畸形等症状。后期则产生落花落果、果实畸形等症状。给我国辣椒和番茄的生产造成了重大的经济损失。
病毒病害的快速检测为病害控制提供了先决条件。目前,最普遍的分子检测手段是聚合酶链式反应(Polymerase Chain Reaction , PCR)。该技术是利用DNA在体外摄氏95°高温时变性会变成单链,低温(经常是60°C左右)时引物与单链按碱基互补配对的原则结合,再调温度至DNA聚合酶最适反应温度(72°C左右),DNA聚合酶沿着磷酸到五碳糖(5'-3')的方向合成互补链。基于聚合酶制造的PCR仪实际就是一个温控设备,能在变性温度,复性温度,延伸温度之间很好地进行控制。利用PCR扩增技术对植物病毒进行检测具有灵敏性和高效性等特点,由于其对任何材料、甚至含量很低的样品都能进行检测,因此被广泛应用于生物学研究的各个领域。
RT-PCR(reverse transcription-Polymerase Chain Reaction , RT-PCR)反转录聚合酶链式反应,在引物的作用下,先对RNA样品进行cDNA合成,在进行扩增。这种方法拥有高灵敏度、强转移性等特点。
PCR扩增或者RT-PCR扩增的首要条件是有一对已知引物。引物的设计涉及到长度、G+C含量、引物自身配对、引物二聚体、3’末端和特异性等情况。
发明内容
本发明的目的在于提供一种检测辣椒脉斑驳病毒专用引物及其检测方法,本发明提供一种检测辣椒脉斑驳病毒所用PCR引物,并以此引物为基础提供了一种龙葵和番茄中辣椒脉斑驳病毒(ChiVMV)的检测方法。
本发明的目的是通过以下技术方案实现的:
一种检测辣椒脉斑驳病毒专用引物,所述一种检测辣椒脉斑驳病毒所用到的PCR引物具体为:
上游引物:ChiVMV-CP-F:如SEQ ID NO.1 所示,即,5’-GCACCATACATTTCAGAAACAGC-3’,
下游引物:ChiVMV-CP-R:如SEQ ID NO.2 所示,即,5’-GAACCACACTGAAGAATATGRATG-3’。
一种检测辣椒脉斑驳病毒方法,所述方法包括以下制备过程:
检测辣椒脉斑驳病毒所用引物在判定辣椒脉斑驳病毒中的应用,用于检测确定龙葵和番茄样品是否受到辣椒脉斑驳病毒(ChiVMV)的侵染;包括如下步骤:
(1)提取待检测龙葵和番茄样品的总RNA,备用;提取方法如下:
采用TRIzol法对植物病叶的总RNA进行提取,实验步骤按照说明书进行:
A.称量花叶重症样品,于液态氮气中研磨至完全粉碎,转移Eppendorf管中;
B. 立即加入TRIzol,室温放置以裂解细胞;离心15 min;
C.取上清液,加入等体积三氯甲烷混合旋涡振荡,室温下静置;离心15 min;
D.取上清液,加入体积异丙醇,混合均匀冷藏;离心15 min;
E.弃上清,用无水乙醇洗涤沉淀;离心15 min;
F.弃上清,室温干燥,加入RNase-free H2O溶解,冰上备用,-80℃冻存;
(2)依据TIANScript RT Kit cDNA(天根生化科技(北京)有限公司)cDNA第一条链合成试剂盒进行反转录;以该cDNA为模板进行PCR扩增;
混合均匀,42℃温浴,反应结束后于95℃下温浴以终止反应,待温度降至室温后加入RNase-Free ddH2O稀释备用;
反应程序为:95℃预变性5min,94℃变性30s,54℃退火40s,72℃延伸40s,实验进行40个循环,72℃终延伸7min;
(3)对(2)中的PCR产物进行琼脂糖凝胶电泳检测,得到阳性条带,目的条带大小预计约为1,000bp,经测序后Blast比对,表明样品中含有辣椒买斑驳病毒;
(4)为进一步确定样品是否受到辣椒脉斑驳病毒侵染,优选情况下,将(3)中待检测PCR产物样品进行回收,回收产物直接与PMD20-T载体进行连接,转化到DH-5α菌株中培养,挑取单菌落进行菌液复苏,并对复苏后的菌液进行菌液PCR初步鉴定,对阳性菌株进行进一步测序,依据测序结果进行比对,进一步确定样品是否被辣椒脉斑驳病毒侵染。
所述的一种检测辣椒脉斑驳病毒方法,所述以该cDNA为模板进行PCR扩增;
cDNA第一条链合成:
2μl M4-T(18);
2μl Super Pure dNTPs(2.5mmol each);
3μl RNA;
补RNase-Free ddH2O至14.5μl;混匀,70℃温浴5min;迅速置于冰上静置2min;
4μl 5×First-Strand buffer;
0.5μl RNase抑制剂(20 U·μL-1);
1μl TIANScript M-MLV反转录酶(200U·L-1)。
所述的一种检测辣椒脉斑驳病毒方法,所述PCR体系设计如下:
cDNA,3μl;
上游引物,1μl;
下游引物,1μl;
dNTP,1μl;
10*Taq Buffer,2.5μl;
Taq,0.2μl;
补灭菌水至25μl。
本发明的优点与效果是:
本发明所提供的PCR引物对ChiVMV的检出具有通用性,不仅能检测出龙葵样品中的ChiVMV,还可以检测到番茄样品中的ChiVMV。在检测过程中利用RT-PCR手段,具有灵敏性高、操作简便等特点。实验表明,利用本发明检测龙葵和番茄上的辣椒脉斑驳病毒(ChiVMV),有较高的特异性。
附图说明
图1为RT-PCR法检测龙葵中辣椒脉斑驳病毒(ChiVMV)的凝胶电泳图。(Matker:DL2000,1~6为病样,7为健康,CK-为水);
图2为RT-PCR法检测番茄中辣椒脉斑驳病毒(ChiVMV)的凝胶电泳图。(Matker:DL2000,1~3为病样,4为健康);
图3 为待检龙葵样品检测出ChiVMV cp基因在NCBI blast比对结果;
图4 为待检番茄样品检测出ChiVMV cp基因在NCBI blast比对结果。
具体实施方式
下面结合实例对本发明做进一步解释说明,在介绍实例之前,首先对本发明中所涉及到的部分物料进行如下简介。
生物材料样品:
待检测样品为疑是被ChiVMV侵染的带有明显被病毒侵染症状的龙葵样品,该样品采自辽宁省沈阳市大东区上园路某小区,其生理症状表现为:叶片黄化、轻微斑驳的症状;
大肠杆菌DH-5α菌株,TIANGEN(天根生化科技(北京)有限公司);
PMD20-T载体,TaKaRa(宝生物工程(大连)有限公司);
上下游引物合成,生工生物工程(上海)股份有限公司合成;
实验仪器及试剂:
凝胶电泳仪,北京六一仪器厂;
TRIzol试剂,TIANGEN(天根生化科技(北京)有限公司);
Taq DNA聚合酶,TIANGEN(天根生化科技(北京)有限公司);
Marker DL2000,TaKaRa(宝生物工程(大连)有限公司);
TIANScript RT Kit试剂盒,TIANGEN(天根生化科技(北京)有限公司)。
实施例1
本实例中所提供的龙葵上辣椒脉斑驳病毒的检测方法,具体包括如下步骤:
(1)提取龙葵植物病叶样品的总RNA,备用;具体操作过程如下:
采用TRIzol法对龙葵病叶的总RNA进行提取,实验步骤按照说明书进行。
A.称量0.2 g龙葵花叶重症样品,于液态氮气中研磨至完全粉碎,转移到1.5mlEppendorf管中;
B.立即加入1ml TRIzol,室温放置10min以裂解细胞;4℃,12000rpm/min 离心15min;
C.取上清液,加入等体积三氯甲烷混合旋涡振荡,室温下静置3min;4℃,12000rpm/min 离心15 min;
D.取上清液,加入0.6倍体积异丙醇,混合均匀冷藏1-2h;4℃,12000rpm/min 离心15 min;
E.弃上清,用1ml 75%无水乙醇洗涤沉淀;4℃,12000rpm/min 离心15 min;
F.弃上清,室温干燥,加入RNase-free H2O溶解,冰上备用,-80℃冻存。
(2)依据TIANScript RT Kit cDNA(天根生化科技(北京)有限公司)cDNA第一条链合成试剂盒对步骤(1)中提取的龙葵植物总RNA进行反转录。以该cDNA为模板进行PCR扩增。
PCR引物设计如下:(该引物特异扩增ChiVMV cp基因序列片段,大小约为1,000bp)
上游引物:ChiVMV-CP-F:如SEQ ID NO.1 所示,即,5’-GCACCATACATTTCAGAAACAGC-3’,
下游引物:ChiVMV-CP-R:如SEQ ID NO.2 所示,即,5’-GAACCACACTGAAGAATATGRATG-3’。
(3)PCR扩增时采用25μl体系,体系设计如下:
步骤(2)中样品的cDNA,3μl;
上游引物,1μl;
下游引物,1μl;
dNTP,1μl;
10*Taq Buffer,2.5μl;
Taq,0.2μl;
补灭菌水至25μl。
反应程序为:95℃预变性5min,94℃变性30s,54℃退火40s,72℃延伸40s,实验进行40个循环,72℃终延伸7min。
PCR产物于4℃冷藏保存待检测,或立即于1%琼脂糖凝胶电泳检测。
(4)取(3)中PCR产物于1%琼脂糖凝胶电泳检测,若样品呈现阳性且条带大小为1,100bp左右,表明样品中含有辣椒脉斑驳病毒。
检测结果如图1所示,阴性对照CK-为水,显阴性,待测样品1~6号呈现阳性,7号健康样品呈现阴性,表明1~6号样品中受到ChiVMV的侵染,健康样品7号中没有收到病毒的侵染。将(3)中待检测PCR产物样品进行回收,回收产物直接与PMD20-T载体进行连接,转化到DH-5α菌株中培养,挑取单菌落进行菌液复苏,并对复苏后的菌液进行菌液PCR初步鉴定,对阳性菌株进行进一步测序,依据测序结果进行比对,测序结果如SEQ IN NO.3所示,与ChiVMV cp基因(GENBANK Accession Nos KF738253, HQ317867, JN692501)进行比对,基本相同,可确定龙葵样品受到ChiVMV的侵染。
总体而言,本发明中通过特异性引物的应用,经过RT-PCR,特定扩增产物的比对,即可快速对植物样品是否受到辣椒脉斑驳病毒的侵染做出判断,该种方法适用于快速、批量化的对样品进行鉴定,而对于不确定是否被该种病毒侵染,或病毒RNA量较低的样品,也可通过连接到PMD20-T载体上,进一步转化、测序进行验证,进而判断该样品是否被辣椒脉斑驳病毒侵染。因此,该种方法鉴定方式简易,样品种类宽泛,为辣椒脉斑驳病毒(ChiVMV)的鉴定提供良好的基础及理论保障。
<110> 沈阳大学
<120> 一种检测辣椒脉斑驳病毒的专用引物及检测方法
<160> 3
<170> DNAMAN 6.0
<210> 1
<211> 23
<212> DNA
<213> PCR引物
<400> 1
gcaccatacatttcagaaacagc
<210> 2
<211> 24
<212> DNA
<213> PCR引物
<400> 2
gaaccacactgaagaatatgratg
<210> 3
<211> 1103
<212> DNA
<213> Chilli veinal mottle virus
<400> 3
gcaccatacatttcagaaacagcactaaagtgtctctacacaagcaaacatggagaggac
gacattggcatataccttaaagcactcattgagggatccaagcaggaagagttggatttc
aatgacagtgaggttactcaccaagcaggagaaagtgttgatgctgggcgcgttaaaggt
gagagcacatcaggaaaacaaacggaccagcaagcattggaaagaaagaacaaaacagaa
ggtcagactcaggcacagtctcgtccaagtgaaatggaagtgccccaggtcagagataga
gatgtcaatgttggaaccacaggaacatttgcagtgccgcgtcttaaaggcatctcttca
aagctgacaataccaaaggttaaaacgaaggccgttgttaacctcgaacaccttttagat
tatgcccctgagcaaatacatataagcaacacaagggcattgcaatcccaatttgcatct
tggtatgaaggtgttaagagtgattatgacgtcacagatgaccagatgcaaataatcttg
aatggtttgatggtttggtgtattgagaatggaacctcaccaaacatcaatggctattgg
gttatgatggatggagatgagcaggttgaatatccgataaaaccactgattgatcatgcc
aaaccatcatttagacaaataatggcacactttagcaacctggctgaagcgtacattgaa
aagcgcaactctgagaagccttatatgccaagatatgggcttcaaagaaaccttaccgat
atgtcattggcgcgatatgcttttgatttttatgaaatgacatcaaaaactcccgttcga
gctcgtgaagcgcatattcaaatgaaggcagctgcattgcgtggcgctagcaatagaatg
tttggactggacggtagggtcggcacacaggaagaggacaccgaaagacacacagcagag
gatgtaaatagaaacatgcacaacctgctgggcgttcgagggatatgatttcttcttcag
cttttaactagtagtaagtggcgtattgtagtatatgtaacttggattatgttgctgatc
atccatattcttcagtgtggttc
Claims (3)
1.一种检测辣椒脉斑驳病毒的专用引物,其特征在于,所述引物的序列具体为:
上游引物:ChiVMV-CP-F:如SEQ ID NO.1 所示,即,5’- GCACCATACATTTCAGAAACAGC-3’,
下游引物:ChiVMV-CP-R:如SEQ ID NO.2 所示,即,5’- GAACCACACTGAAGAATATGRATG-3’,
所述的引物PCR扩增的产物如SEQ ID NO.3所示。
2.一种检测辣椒脉斑驳病毒方法,其特征在于,所述方法包括如下步骤:
(1)提取待检测龙葵和番茄样品的总RNA,备用;总RNA的提取方法如下:
采用TRIzol法对植物病叶的总RNA进行提取,实验步骤按照说明书进行:
称量花叶重症样品,于液态氮气中研磨至完全粉碎,转移Eppendorf管中;
立即加入TRIzol,室温放置以裂解细胞;离心15 min;
取上清液,加入等体积三氯甲烷混合旋涡振荡,室温下静置;离心15 min;
取上清液,加入体积异丙醇,混合均匀冷藏;离心15 min;
弃上清,用无水乙醇洗涤沉淀;离心15 min;
弃上清,室温干燥,加入RNase-free H2O溶解,冰上备用,-80℃冻存;
(2)依据TIANScript RT Kit cDNA第一条链合成试剂盒进行反转录;以cDNA为模板进行PCR扩增;
混合均匀,42℃温浴,反应结束后于95℃下温浴以终止反应,待温度降至室温后加入RNase-Free ddH2O稀释备用;
其中,PCR引物如权利要求1所示;
PCR扩增时采用25μL体系,体系设计如下:
cDNA,3μl;
上游引物,1μl;
下游引物,1μl;
dNTP,1μl;
10*Taq Buffer,2.5μl;
Taq,0.2μl;
补灭菌水至25μl;
反应程序为:95℃预变性5min,94℃变性30s,54℃退火40s,72℃延伸40s,实验进行40个循环,72℃终延伸7min;
(3)对(2)中的PCR产物进行琼脂糖凝胶电泳检测,得到阳性条带,目的条带大小为1,103bp,经测序后进行Blast比对,表明样品中含有辣椒脉斑驳病毒;
(4)为进一步确定样品是否受到辣椒脉斑驳病毒侵染,将(3)中待检测PCR 产物样品进行回收,回收产物直接与PMD20-T载体进行连接,转化到DH-5α菌株中培养,挑取单菌落进行菌液复苏,并对复苏后的菌液进行菌液PCR初步鉴定,对阳性菌株进行进一步测序,依据测序结果进行比对,进一步确定样品是否被辣椒脉斑驳病毒侵染。
3.根据权利要求2所述的一种检测辣椒脉斑驳病毒方法,其特征在于,
cDNA第一条链合成的体系如下:
2μl M4-T18;
2μl Super Pure dNTPs,其浓度为2.5mmol;
3μl RNA;
补RNase-Free ddH2O至14.5μl;
4μl 5×First-Strand buffer;
0.5μl RNase抑制剂,其浓度为20 U•μL-1;
1μl TIANScript M-MLV反转录酶,其浓度为200U•L-1。
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