CN108842000A - 用于鉴别DAdV-3和DAdV-A的引物组 - Google Patents
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Abstract
本发明提供用于鉴别DAdV‑3和DAdV‑A的引物组,属于鸭病学研究领域。采用了如SEQ ID NO.1‑3所示的引物组(3条引物),建立能够对我国鸭群中出现的两种鸭腺病毒(DAdV‑3和DAdV‑A)的鉴别诊断方法,本发明基于DAdV‑3和DAdV‑A的Hexon基因特点,建立一种仅需一套引物组(3条引物),即可科学对DAdV‑3和DAdV‑A进行鉴别,为在鸭群中开展腺病毒分子流行病毒学调查和科学防控相关疫病提供支撑,具有十分重要的研究意义。
Description
技术领域
本发明属于鸭病学研究领域,具体涉及用于鉴别DAdV-3和DAdV-A的引物组。
背景技术
腺病毒颗粒为无囊膜、核衣壳呈二十面体对称、线性、双链DNA病毒,其基因组全长约为33kd左右。其中,六邻体蛋白(Hexon)是腺病毒科各属病毒最主要的结构蛋白,每个Hexon蛋白表面携带有病毒主要的属的种特异性抗原决定簇,是病毒中和抗体的靶目标,含有病毒主要的保护性抗原基因簇,与病毒的致病性密切相关。
根据国际病毒分类委员会(International Committee on Taxonomy ofViruses,ICTV)的最新分类(https://talk.ictvonline.org/),腺病毒科(Family:Adenoviridae)下设5个属(Genera),分别为富AT腺病毒属(Genus: Atadenovirus)、禽腺病毒属(Genus: Aviadenovirus)、鱼腺病毒属(Genus: Ichtadenovirus)、哺乳动物腺病毒属(Genus: Mastadenovirus)和唾液酸酶病毒属(Genus: Siadenovirus)。
研究发现,禽类中存在多种腺病毒感染,其中感染鸡的腺病毒有5种:禽腺病毒A型(Fowl aviadenovirus A,FAdV-A)、禽腺病毒B型(Fowl aviadenovirus B,FAdV-B)、禽腺病毒C型(Fowl aviadenovirus C,FAdV-C)、禽腺病毒D型(Fowl aviadenovirus D,FAdV-D)、禽腺病毒E型(Fowl aviadenovirus E,FAdV-E),均属于禽腺病毒属。感染火鸡(Turkey)的腺病毒有4种,分别属于腺病毒科的两个属,其中火鸡腺病毒B型(Turkey aviadenovirusB)、火鸡腺病毒C型(Turkey aviadenovirus C)和火鸡腺病毒D型(Turkey aviadenovirusD)属于禽腺病毒属;而火鸡唾液酸酶病毒A型(Turkey siadenovirus A)属于唾液酸酶病毒属。感染鹦鹉(Psittacine)的腺病毒有2种,分别属于腺病毒科的两个属,其中鹦鹉富AT腺病毒属A型(Psittacine atadenovirus A)属于富AT腺病毒属;而鹦鹉腺病毒B型(Psittacine aviadenovirus B)属于禽腺病毒属。
近年来,随着我国水禽(尤其是鸭)饲养规模的不断扩大,水禽中新发疫病也越来越多、越来越复杂。鸭群中已报道的腺病毒有属于富AT腺病毒属的鸭腺病毒A型(duckadenovirus A,缩写为DAdV-A)。2014年,随着高通量技术的发展,Marek A等测定了1株鸭源腺病毒(GR株,GenBank登录号NC024486)全基因组序列,并将其命名为鸭腺病毒2型(duckadenovirus 2),属于禽腺病毒属新成员(Marek A, Kaján GL, Kosiol C, et al.Complete genome sequences of pigeon adenovirus 1 and duck adenovirus 2 extendthe number of species within the genus Aviadenovirus[J]. Virology, 2014, 462-463: 107-114.),随后ICTV将其科学命名为鸭腺病毒B型(duck aviadenovirus B,用来区别鸭腺病毒A型)。2014年以来,在我国番鸭中出现一种新的鸭腺病毒(CH-GD-2014株,GenBank登录号KR135164),该病毒和鸭腺病毒2型(duck adenovirus 2)GR株存在以下明显区别:①其全基因组序列和GR株的核苷酸同源性仅为92.3%;②CH-GD-2014株和GR株的Hexon基因的核苷酸同源性仅为76.6%,低于80.0%;③CH-GD-2014株有2个纤突蛋白(Fiber1、Fiber 2),而GR株只有1个纤突蛋白(Fiber)。基于上述特征表明在我国鸭群中流行一种新的鸭腺病毒,并将其命名为鸭腺病毒3型(duck adenovirus 3,DAdV-3)(周镇海.鸭腺病毒3型基因组序列分析及致病性研究[D].华南农业大学硕士论文,2016.)。
多重PCR(multiplex PCR),又称多重引物PCR或复合PCR,它是在同一PCR反应体系里加上二对或以上引物,同时扩增出多个核酸片段的PCR反应,其反应原理,反应试剂和操作过程与一般PCR相同,灵敏度与一般PCR相当。由于其具有高效性、系统性和经济简便性被广泛应用于多种病原微生物的同时检测或鉴定某些病原微生物。一般而言,对两个病原进行鉴别诊断,需要针对2个病原的分别设计引物(即共需要4条引物),来建立鉴别诊断方法。本研究基于DAdV-3和DAdV-A的Hexon基因特点,建立一种仅需一套引物组(3条引物),即可科学对DAdV-3和DAdV-A进行鉴别,为鸭群中开展腺病毒感染类型的流行病学调查及科学防控相关疾病奠定基础,具有十分重要的研究意义。
发明内容
本发明的目的在于提供用于鉴别DAdV-3和DAdV-A的引物组,建立一种仅需一套引物组(3条引物),即可科学对DAdV-3和DAdV-A进行鉴别,为鸭群中开展腺病毒感染类型的流行病学调查及科学防控相关疾病奠定基础。
为实现上述目的采用以下技术方案:
用于鉴别DAdV-3和DAdV-A的引物组,所述引物组如下:
DAdV-3A-F:5’-TGGCACTACAAGACAGAA-3’,
DAdV-3-R:5’-AGTTCTCCACCAGGTTGAAAG -3’,
DAdV-A-R2:5’- TATCCCCAGCAACCAGTCCAG -3’。
按照DreamTaq Green PCR Master Mix(2×)试剂盒推荐的的50μL体系进行扩增,其中2×PCR Master Mix扩增反应液25 μL、引物DAdV-3A-F(10μM)2.0 μL、引物DAdV-3-R(10μM)1.0 μL、引物DAdV-A-R2(10μM)1.0 μL、提取的核酸模板1.0 μL、补充灭菌去离子水至终体积50μL,混匀后进行PCR扩增。
扩增条件为95 ℃预变性3 min后进入循环,95 ℃变性30 s 、△T(52 ℃-62 ℃)退火30 s、72 ℃延伸50s,35个循环结束后,72℃终延伸10 min,反应结束后按照常规琼脂糖凝胶电泳鉴定。△T(52 ℃-62 ℃)表示退火温度在此区间进行优化,经优化后的最佳退火温度为55 ℃。
本发明的另一目的是提供所述的引物组在制备检测DAdV-3和DAdV-A试剂盒中的应用。
本发明的优点在于:
本发明基于DAdV-3和DAdV-A的Hexon基因特点,建立一种仅需一套引物组(3条引物)的PCR方法,对DAdV-3最低检测限为3.24×101pg;DAdV-A最低检测限为5.09×101pg;本发明方法简单实用、方便快捷,即可科学对DAdV-3和DAdV-A进行鉴别,为鸭群中开展腺病毒感染类型的流行病学调查及科学防控相关疾病奠定基础,具有十分重要的研究意义。
附图说明
图1 DAdV-3和DAdV-A上游引物设计区。
图2 DAdV-3下游引物设计区。
图3 DAdV-A下游引物设计区。
图4 PCR产物的电泳;其中M:DNA分子量标准;1:DAdV-A;2:DAdV-3;3:DAdV-3和DAdV-A混合感染;4:DEV;5:MDPV;6:DuCV;7:GPV;8:E. coli;9:R.A.;10:P.M.;11:DAdV-2。
图5 DAdV-3灵敏性试验;其中M:DNA分子量标准;1-6:3.24×105-3.24×100pg连续稀释浓度模板。
图6 DAdV-A灵敏性试验;其中M:DNA分子量标准;1-6:5.09×105-5.09×100pg连续稀释浓度模板。
具体实施方式
实施例1
1、材料
1.1 毒株和菌株
试验用病原DAdV-3(W1FJ株,GenBank登录号MH349774)、DAdV-A(FJ12025株,GenBank登录号KF286430)由福建省农业科学院畜牧兽医研究所保存。
试验用对照毒株鸭肠炎病毒(DEV)、番鸭细小病毒(MDPV)、鸭圆环病毒(DuCV)、鸭源鹅细小病毒(GPV);试验用对照菌株鸭大肠杆菌(E. coli)、鸭疫里氏杆菌(R.A.)和鸭源禽多杀性巴氏杆菌(P.M.),均由福建省农业科学院畜牧兽医研究所保存。鸭腺病毒2型(DAdV-2)的Hexon基因(GR株,GenBank登录号NC024486)序列全长由生工生物工程(上海)股份有限公司合成。
引物设计
根据美国国家生物技术信息中心(National Center of BiotechnologyInformation,NCBI)数据库GenBank上腺病毒科DAdV-3代表株:CH-GD-2014株(KR135164)、W1GD株(MH349773)、W1FJ株(MH349774)、W1AH株(MH349775)和DAdV-A代表株:E05株(EF532411)、FJ12025株(KF286430)、D11-JW-012株(KJ452170)、D11-JW-017株(KJ452171)的Hexon基因,利用Lasergene DNAStar MegAlign进行核苷酸系列分析比对,确定DAdV-3和DAdV-A基因保守区作为上游通用引物设计区(图1);确定DAdV-3保守但和DAdV-A基因差异较大的差异区作为DAdV-3下游引物设计区(图2);确定DAdV-A保守但和DAdV-3基因差异较大的差异区作为DAdV-A下游引物设计区(图3)。利用引物设计软件Oligo 7.0设计一套引物组,如下:
DAdV-3A-F:5’- CATACMGYGGMACAGCTTACAATC -3’,
DAdV-3-R:5’-AGTTCTCCACCAGGTTGAAAG -3’,
DAdV-A-R2:5’- TATCCCCAGCAACCAGTCCAG -3’。
主要试剂
DreamTaq Green PCR Master Mix(2×)购自Thermo Scientific公司,EasyPureGenomic DNA Kit、EasyPure Bacteria Genomic DNA Kit均购自北京全式金生物技术有限公司。
试验方法的建立
2.1 基因组DNA的提取
DAdV-3、DAdV-A、DEV、MDPV、DuCV、GPV按照EasyPure Genomic DNA Kit试剂盒的方法提取相应的基因组DNA,-80℃冻存备用。
E. coli、R.A.和P.M.按照 EasyPure Bacteria Genomic DNA Kit试剂盒的方法提取相应的基因组DNA,-80℃冻存备用。
2.2 反应液的配置和退火温度的优化
按照DreamTaq Green PCR Master Mix(2×)试剂盒推荐的的50μL体系进行扩增,其中2×PCR Master Mix扩增反应液25 μL、引物DAdV-3A-F(10μM)2.0 μL、引物DAdV-3-R(10μM)1.0 μL、引物DAdV-A-R2(10μM)1.0 μL、提取的核酸模板(DAdV-3、DAdV-A、DAdV-3和DAdV-A混合)各1.0 μL、补充灭菌去离子水至终体积50μL,混匀后进行PCR扩增。
扩增条件为95 ℃预变性3 min后进入循环,95 ℃变性30 s 、△T(52 ℃-62 ℃)退火30 s、72 ℃延伸50s,35个循环结束后,72℃终延伸10 min,反应结束后按照常规琼脂糖凝胶电泳鉴定。△T(52 ℃-62 ℃)表示退火温度在此区间进行优化。经优化后的最佳退火温度为55 ℃。
结果可见(图4),当模板仅加入DAdV-3时,出现大小为814bp的条带(第2泳道);当模板仅加入DAdV-A时,出现大小为420bp的条带(第1泳道);当模板加入DAdV-3和DAdV-A混合时,出现大小为814bp和420bp的两条带(第3泳道)。
2.3 特异性试验
将优化后的双重PCR体系和条件,对DEV、MDPV、DuCV、GPV、E. coli、R.A.和P.M.以及DAdV-2对照进行扩增,均未见扩增条带。结果见图4,DEV(第4泳道)、MDPV(第5泳道)、DuCV(第6泳道)、GPV(第7泳道)、E. coli(第8泳道)、R.A.(第9泳道)和P.M.(第10泳道)以及DAdV-2(第11泳道),表明建立的方法特异性强,对常见水禽病原均无交叉反应。
2.4敏感性试验
将提取的DAdV-3和DAdV-A基因组DNA,测定OD值后,进行连续10被稀释。其中DAdV-3浓度分别为3.24×105-3.24×100pg;DAdV-A浓度分别为5.09×105-5.09×100pg。结果可见,DAdV-3最低检测限为3.24×101pg(图5);DAdV-A最低检测限为5.09×101pg(图6),和常规PCR相当。
临床试验
对49份临床送检鸭源病料按照常规方法研磨处理后,利用EasyPure Genomic DNA Kit提取相应的核酸DNA,利用优化后的PCR方法进行DAdV-3和DAdV-A感染的检测。结果可见,检测到9份DAdV-3感染阳性,阳性率为18.37%;检测到5份DAdV-A感染阳性,阳性率为10.20%;检测到2份DAdV-3和DAdV-A共感染阳性,阳性率为4.08%。
将PCR反应结束后的阳性目的片段利用琼脂糖凝胶回收试剂盒(DP209,天根生化科技(北京)有限公司)分别进行切胶回收。按照pEASY-T1 Simple Cloning Kit克隆连接试剂盒(CT111-01,北京全式金生物技术有限公司)说明书将Hexon基因片段克隆到pEASY-T1载体上,随机挑取8个单菌落于氨苄青霉素(含量为100 μg/mL)抗性的LB液体培养基培养14h后,利用快速质粒小提试剂盒(DP105,天根生化科技(北京)有限公司)提取相应的质粒。采用PCR扩增时的引物和条件对提取的质粒分别进行PCR鉴定,将筛选出的阳性重组质粒送宝日医生物技术(北京)有限公司进行测序。将测序结果在NCBI上进行BLAST分析验证,均为相应的DAdV-3和DAdV-A的Hexon基因片段,其中DAdV-3阳性片段和DAdV-3(W1FJ株,GenBank登录号MH349774)的Hexon基因同源性在99.0%以上;DAdV-A阳性片段和DAdV-A(FJ12025株,GenBank登录号KF286430)的Hexon基因同源性在99.0%以上。上述测序结果和PCR检测结果的符合率均为100%。
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。
SEQUENCE LISTING
<110> 福建省农业科学院畜牧兽医研究所
<120> 用于鉴别DAdV-3和DAdV-A的引物组
<130> 3
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 18
<212> DNA
<213> 人工序列
<400> 1
tggcactaca agacagaa 18
<210> 2
<211> 21
<212> DNA
<213> 人工序列
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agttctccac caggttgaaa g 21
<210> 3
<211> 21
<212> DNA
<213> 人工序列
<400> 3
tatccccagc aaccagtcca g 21
Claims (2)
1.用于鉴别DAdV-3和DAdV-A的引物组,其特征在于:所述引物组如下:
DAdV-3A-F:5’-TGGCACTACAAGACAGAA-3’,
DAdV-3-R:5’-AGTTCTCCACCAGGTTGAAAG -3’,
DAdV-A-R2:5’- TATCCCCAGCAACCAGTCCAG -3’。
2.权利要求1所述的引物组在制备检测DAdV-3和DAdV-A试剂盒中的应用。
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