CN102559939B - 鉴别71型和非71型肠道病毒的单管二重一步法rt-pcr引物及其应用 - Google Patents
鉴别71型和非71型肠道病毒的单管二重一步法rt-pcr引物及其应用 Download PDFInfo
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Abstract
鉴别71型和非71型肠道病毒的单管二重一步法RT-PCR引物及其应用。本发明提供的二重一步法RT-PCR引物包括肠道病毒通用引物TACTTCGAGAARCCTAGTAWCACC和ACGGACACCCAAAGTAGTCGGTTC,以及肠道病毒71型(EV71)特异性引物CCCAACACAGCYTAYATAATAGC和GTAYCCAYGCCCTGACGTGYTTC。通用引物用于扩增所有肠道病毒5’UTR基因,特异性引物用于扩增所有EV71VP1基因。本发明的优点:(1)两对引物同时在一个PCR管反应,快速简便,节省成本;(2)可早期鉴别EV71和非EV71型引起的手足口病;(3)检测灵敏度高,特异性好;(4)普通RT-PCR仪就能操作,易于推广。
Description
技术领域
本发明属于病毒核酸检测领域,涉及鉴别71型和非71型肠道病毒的单管二重一步法RT-PCR引物及其应用。
背景技术
手足口病(hand foot and mouth disease,HFMD)是由肠道病毒引起的急性传染病,主要发生于3岁以下儿童,临床表现为手、足、口腔等部位的疱疹。而引起手足口病的病原体有多种,如柯萨奇病毒A组的16、4、6、10、12,B组2、3、5,埃可病毒4、19、30,以及肠道病毒 71型(Enterovirus 71,EV71)等。其中以EV71和柯萨奇病毒A16(Coxsackievirus 16, CA16)最为常见。通常情况下,EV71和CA16等非EV71感染引起的手足口病在临床症状和体征方面难以区别,但EV71感染除了引起手足口病外,部分患者还能出现无菌性脑膜炎、脑炎、脊髓灰质炎样的麻痹和神经源性肺水肿等多种神经系统并发症,病情凶险,可严重危害婴幼儿的生命健康,故倍受重视。近年来,我国及周边地区手足口病疫情日趋严重。2011年我国全年累计报告的病例161万余例,死亡509例。因此早期、快速、简便、准确地鉴别EV71型和非EV71型手足口病,对于积极的临床救治和疫情防控具有重要意义。
传统的病原学诊断采用细胞培养分离和鉴定病毒的手段,耗时且操作复杂,无法满足病毒流行期间同时处理大量样本的需要。分子生物学的进展开拓了肠道病毒的快速诊断技术,特别是PCR技术,由于其检测材料用量少,快速,灵敏度高,特异性好,在病毒感染的诊断中发挥越来越多的重要作用。今RT-PCR技术已成为手足口病病原体快速诊断的重要手段,但目前建立的方法主要针对EV71和CA16,很容易造成其它病原体的漏诊;且每个检测都要单独进行,繁琐费时;若要检测其他EV,还要再增加一个PCR反应管,试剂消耗大,操作步骤多,容易造成实验失败。如能建立一种单管二重一步法RT-PCR,鉴别EV71型和非EV71型手足口病,将具有广泛的应用前景。
发明内容
解决的技术问题:本发明针对手足口病病原学检查方法的单一性,耗时耗力的不足,提供一种高效、便捷的鉴别71型和非71型肠道病毒的单管二重一步法RT-PCR引物及其应用。
技术方案:
鉴别71型和非71型肠道病毒的单管二重一步法RT-PCR引物,同时包括肠道病毒5’UTR通用引物和EV71 VP1基因特异性引物,所述肠道病毒5’UTR通用引物为:
引物F2:5’-TACTTCGAGAARCCTAGTAWCACC-3’ ;
引物R1:5’-ACGGACACCCAAAGTAGTCGGTTC-3’ ;
所述EV71 VP1基因特异性引物为:
引物Fa: 5’-CCCAACACAGCYTAYATAATAGC-3’ ;
引物Rb:5’-GTAYCCAYGCCCTGACGTGYTTC -3’;
其中R=A或G,W= A或T,Y=C或T。
上述引物是在相应引物上从3’末端、从5’末端或者同时从3’和5’末端有1-5个附加核苷酸加入或缺失的引物。
上述引物在制备鉴别EV71型和非EV71型手足口病核酸检测试剂中的应用。
从Genbank下载了世界各地的肠道病毒毒株,包含了肠道病毒所有67个血清型,用DNAMAN软件进行序列比对:
依据肠道病毒5’UTR设计通用引物:
引物F1:5’- AMCAAGCACTTCTGTTWCCCCGG -3’ 23bp
引物F2: 5’- TACTTCGAGAARCCTAGTAWCACC -3’ 24bp
引物R1:5’- ACGGACACCCAAAGTAGTCGGTTC -3’ 24bp
引物R2: 5’- CTYGATTGTCACCATAAGCAGCCA -3’ 24bp;
依据所有EV71病毒VP1基因设计特异性引物:
引物Fa: 5’- CCCAACACAGCYTAYATAATAGC -3’ 23bp
引物Fb: 5’- CAGGGAGATAGRGTGGCRGATG -3’ 22bp
引物Fc: 5’- TGGRGCATCRTCRAATRCTAGTG -3’ 23bp
引物Ra: 5’- CGCACYGAGAAMGTGCCCATC -3’ 21bp
引物Rb: 5’- GTAYCCAYGCCCTGACGTGYTTC -3’ 23bp。
所述的引物变体是指在相应引物上从3’末端,从5’末端或者从3’和5’末端有1-5个附加核苷酸加入或缺失的引物。其中M=A或C,R=A或G,W= A或T,Y=C或T。
通过肠道病毒通用引物不同组合进行RT-PCR,筛选出扩增效率高的引物F1+R1、F1+R2、F2+R1、F2+R2;通过EV71特异性引物不同组合进行RT-PCR,筛选出扩增效率高的引物Fa+Rb、Fb+Rb、Fc+Ra、Fc+Rb。然后用F1+R1、F1+R2、F2+R1、F2+R2分别与Fa+Rb、Fb+Rb、Fc+Ra、Fc+Rb配对进行RT-PCR,初步筛选出8对候选二重引物F1R1FaRb、F1R1FbRb、F1R1FcRb、F1R2FaRb、F2R1FaRb、F2R1FbRb、F2R1FcRb、F2R2FaRb。随后用0.1、0.01、0.001 TCID50/mL EV71-NJ1001检测上述候选二重引物RT-PCR的敏感性,最终筛选出本发明的二重引物F2R1FaRb。
一种用于鉴别71型和非71型肠道病毒的单管二重一步法RT-PCR的应用,采用上述筛选的二重引物对EV71和非EV71肠道病毒相关基因进行扩增。
本发明进行单管二重一步法RT-PCR扩增时,采用QIAamp Viral RNA Mini Kit (QIAGEN)提取病毒RNA,用One-Step RT-PCR Kit (QIAGEN)扩增,扩增体系如下:
H2O (RNase-free) | 7.5 μL |
5×One-Step RT-PCR Buffer | 4.0 μL |
dNTP Mix(10 mM) | 0.8 μL |
F2(10 μM) | 0.6 μL |
R1(10 μM) | 0.6 μL |
Fa(10 μM) | 0.35μL |
Rb(10 μM) | 0.35μL |
One-Step RT-PCR Enzyme Mix | 0.8 μL |
Template RNA | 5.0 μL |
Total | 20.0 μL |
本发明进行单管二重一步法RT-PCR扩增的反应条件为:50℃逆转录45min,95℃变性15min,进入循环:94℃变性30s,50℃退火30s,72℃延伸80s,共50个循环,末次循环后72℃延伸10min。
如上所述为二重RT-PCR引物在鉴别EV71型与非EV71型手足口病的方法。
有益效果:应用本技术方案所公开的引物,可用于制备鉴别EV71型与非EV71型手足口病的检测试剂,该检测试剂敏感性好、特异性高,能实现一次性完成EV71型与非EV71型肠道病毒相关基因的扩增,从而显著提高EV71型与非EV71型手足口病的检测效率。本发明所需耗材和试剂用量显著少于传统PCR扩增方法,节省费用;而工作量与一次性普通RT-PCR相当,减少各种操作失误发生的可能,提高检测的成功率。
附图说明
图1为肠道病毒通用引物不同组合与EV71特异性引物不同组合同时对EV71-1001进行RT-PCR。1-4为F1+R1、F1+R2、F2+R1、F2+R2;5-10为Fa+Ra、Fa+Rb、Fb+Ra、Fb+Rb、Fc+Ra、Fc+Rb。
图2为肠道病毒通用引物与EV71特异性引物组成的16对二重引物对EV71-1001进行RT-PCR。1:F1 R1FaRb、2:F1R1FbRb、3:F1R1FcRa、4:F1R1FcRb、5:F1R2FaRb、6:F1R2FbRb、7:F1R2FcRa、8:F1R2FcRb、9:F2R1FaRb、10:F2R1FbRb、11:F2R1FcRa、12:F2R1FcRb、13:F2R2FaRb、14:F2R2FbRb、15:F2R2FcRa、16:F2R2FcRb。
图3为分别用浓度为0.1、0.01、0.001 TCID50/mL EV71-1001病毒液去检测候选二重引物RT-PCR的敏感性。1-3:F1R1FaRb、4-6:F1R1FbRb、7-9:F1R1 FcRb、10-12:F1R2FaRb、13 -15:F2R1FaRb、16-18:F2R1FbRb、19- 21:F2R1FcRb、22-24:F2R2FaRb。
图4为单对引物RT-PCR与单管二重一步法RT-PCR敏感性检测。A为EV71-1001毒株6种不同浓度的病毒液,即10、1、0.1、0.01、0.001、0.0001TCID50/mL。1-6:单对肠道病毒通用引物F2与R1扩增以上6种不同浓度的病毒液(以下类似);7-12:单对EV71特异性引物Fa和Rb扩增;13-18:二重RT-PCR引物F2R1FaRb扩增;
图5为单对引物RT-PCR与单管二重一步法RT-PCR敏感性检测。图5B为CA16-1001 6毒株6种不同浓度的病毒液,即10 、1、0.1、0.01、0.001、0.0001TCID50/mL。1-6:单对肠道病毒通用引物F2与R1扩增;7-12:二重RT-PCR引物F2R1FaRb扩增。图5C为Echo6-1001毒株5种不同浓度的病毒液,即10 、1、0.1、0.01、0.001 TCID50/mL。1-5:单对肠道病毒通用引物F2与R1扩增;6-10:二重RT-PCR引物F2R1FaRb扩增;
图6为实施例中本发明单管二重一步法RT-PCR特异性检测。1-5:EV71-1001、EV71-1002、EV71-1003、EV71-1004、EV71-1005;6-9:CA16-1001、CA16-1002、CA16-1003、CA16-1004;10:Echo 6-1001。
具体实施方式
以下实施例用于说明本发明,但不用来限制本发明的保护范围。本发明涉及到的毒株EV71-1001、EV71-1002、EV71-1003、EV71-1004、EV71-1005、CA16-1001、CA16-1002、CA16-1003、CA16-1004、埃克病毒6型(Echo6-1001)均由本实验室从南京市儿童医院感染科手足口病患儿临床标本中分离并经间接免疫荧光检测、VP1基因测序等方法鉴定。
实施例1:
(一)引物
从Genbank下载了肠道病毒所有67个血清型的216个基因序列,用DNAMAN软件进行序列比对,依据肠道病毒基因保守序列5’UTR设计通用引物F1 、F2、 R1、 R2,依据所有EV71病毒VP1基因保守序列设计特异性引物Fa、Fb、Fc、Ra、Rb。
肠道病毒通用引物:
引物F1:5’- AMCAAGCACTTCTGTTWCCCCGG -3’ 23bp
引物F2: 5’- TACTTCGAGAARCCTAGTAWCACC -3’ 24bp
引物R1:5’- ACGGACACCCAAAGTAGTCGGTTC -3’ 24bp
引物R2: 5’- CTYGATTGTCACCATAAGCAGCCA -3’ 24bp;
依据所有EV71病毒VP1基因设计特异性引物:
引物Fa: 5’- CCCAACACAGCYTAYATAATAGC -3’ 23bp
引物Fb: 5’- CAGGGAGATAGRGTGGCRGATG -3’ 22bp
引物Fc: 5’- TGGRGCATCRTCRAATRCTAGTG -3’ 23bp
引物Ra: 5’- CGCACYGAGAAMGTGCCCATC -3’ 21bp
引物Rb: 5’- GTAYCCAYGCCCTGACGTGYTTC -3’ 23bp。
通过肠道病毒通用引物不同的组合进行RT-PCR,筛选出扩增效率高的引物F1+R1、F1+R2、F2+R1、F2+R2;通过EV71特异性引物不同的组合进行RT-PCR,筛选出扩增效率高的引物Fa+Rb、Fb+Rb、Fc+Ra、Fc+Rb(图1)。然后用F1+R1、F1+R2、F2+R1、F2+R2分别与Fa+Rb、Fb+Rb、Fc+Ra、Fc+Rb组合进行RT-PCR,初步筛选出8对候选二重引物F1R1FaRb、F1R1FbRb、F1R1FcRb、F1R2FaRb、F2R1FaRb、F2R1FbRb、F2R1FcRb、F2R2FaRb(图2)。随后用0.1、0.01、0.001 TCID50/mL EV71-1001检测上述候选二重引物RT-PCR的敏感性,最终筛选出本发明的二重引物F2R1FaRb(图3)。
(二)单管二重一步法RT-PCR
1、样本的制备及核酸的抽提
咽拭子标本:采集发病一周内的患儿咽拭子标本,用专用采样棉签时,在咽后壁和两侧扁桃体部位适度拭抹后将棉签装入2mL病毒保存液中,充分震荡混匀后取140mL悬液,采用QIAamp Viral RNA Mini Kit (QIAGEN)提取病毒RNA;
病毒液标本:将EV71、CA16和Echo6病毒液接种至单层人恶性胚胎横纹肌瘤RD细胞上,36℃、5%vt CO2培养,逐日观察细胞病变效应(CPE),当CPE达80%以上后收获病毒,冻融三次后12000rpm离心后取140mL上清,采用QIAamp Viral RNA Mini Kit (QIAGEN)提取病毒RNA,其中EV71-1001、CA16-1001和Echo6-1001通过微量细胞培养法计算病毒滴度,使用DMEM培养基将病毒进行梯度稀释,得到不同浓度的病毒液用于单对引物RT-PCR和单管二重一步法RT-PCR敏感性检测。
2、单管二重一步法RT-PCR
反应体系:
H2O (RNase-free) | 7.5 μL |
5×One-Step RT-PCR Buffer | 4.0 μL |
dNTP Mix(10 mM) | 0.8 μL |
F2(10 μM) | 0.6 μL |
R1(10 μM) | 0.6 μL |
Fa(10 μM) | 0.35μL |
Rb(10 μM) | 0.35μL |
One-Step RT-PCR Enzyme Mix | 0.8 μL |
Template RNA | 5.0 μL |
Total | 20.0 μL |
扩增条件:
50℃逆转录45min,95℃变性15min,进入循环:94℃变性30s,50℃退火30s,72℃延伸80s,共50个循环,末次循环后72℃延伸10min。
(三)PCR扩增产物的电泳鉴定
取PCR扩增产物5μL,加1μL溴酚蓝上样缓冲液,混匀,点样于20g/L琼脂糖凝胶(含0.5μL/mL溴化乙锭),以100bpDNA分子量Marker作为标准分子参照,在5V/cm的电场强度下于1倍的TAE电泳缓冲液电泳40min,凝胶成像系统紫外线下观察结果并拍照。实验结果表明本发明方法针对EV71的最低检测限为0.001 TCID50/mL,对非EV71的最低检测限为0.01 TCID50/mL(图4-5)。同时对所有的EV71毒株均能扩增出306bp与889pb两条目的条带,而对非EV71毒株均扩增出一条306bp目的条带(图6)。
3、本发明方法的临床应用
使用本发明的方法对165份来源于手足口病患者的咽拭子标本进行单管二重一步法RT-PCR检测,手足口病检测阳性有113份,阳性率达68.5%,其中EV71阳性标本76份,非EV71阳性标本有37份。从76份EV71阳性PCR产物中随机选取49份对VP1基因测序,结果表明49份均为EV71;从37份非EV71阳性PCR产物中随机选取24份对5’UTR基因测序,结果为:9份CA16、8份CA10、2份CA6、2份Echo-6、2份Echo-9和1份CA12。以上结果表明本发明方法的特异性达100%。结合上述敏感性试验结果,本发明具有敏感性高,特异性好,操作简便,节省成本等明显优势。
序列表
<110> 东南大学
<120> 鉴别71型和非71型肠道病毒的单管二重一步法RT-PCR引物及其应用
<130>
<160> 9
<170> PatentIn version 3.3
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Claims (2)
1.鉴别71型和非71型肠道病毒的单管二重一步法RT-PCR引物,其特征在于同时包括肠道病毒5’UTR通用引物和EV71 VP1基因特异性引物,所述肠道病毒5’UTR通用引物为:
引物F2:5’-TACTTCGAGAARCCTAGTAWCACC-3’ ;
引物R1:5’-ACGGACACCCAAAGTAGTCGGTTC-3’ ;
所述EV71 VP1基因特异性引物为:
引物Fa: 5’-CCCAACACAGCYTAYATAATAGC-3’ ;
引物Rb:5’-GTAYCCAYGCCCTGACGTGYTTC -3’;
其中R=A或G,W= A或T,Y=C或T。
2.权利要求1所述引物在制备鉴别EV71型和非EV71型手足口病核酸检测试剂中的应用。
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