CN116694826A - 一种同时检测wssv、ihhnv、ehp和nhp的多重pcr引物探针组合及方法 - Google Patents
一种同时检测wssv、ihhnv、ehp和nhp的多重pcr引物探针组合及方法 Download PDFInfo
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Abstract
本发明公开了一种同时检测WSSV、IHHNV、EHP和NHP的多重PCR引物探针组合及方法。该多重PCR引物探针组合的具体序列如SEQ ID NO.1~12所示。本发明提供的基于该组引物和试剂盒的检测方法具有操作简单、灵敏度高、特异性强、检测速度快的优点,能够同时检测对虾WSSV、IHHNV、EHP和NHP这四种病原菌,大大节约检测时间。
Description
技术领域
本发明属于分子生物学技术领域,具体涉及一种同时检测WSSV、IHHNV、EHP和NHP的多重PCR引物探针组合及方法和用途。
背景技术
白斑综合征是由白斑综合征病毒WSSV引起的一种疫病,该病被世界动物卫生组织(OIE)要求强制通报。发病急、死亡率高,死亡速度快是该病的明显特点,WSSV在自然环境中的传播形式主要有垂直和水平两种途径。二十多年来该病一直是全球对虾养殖产业的主要威胁。WSSV的宿主范围非常宽泛,迄今为止已经确定了WSD的98个潜在宿主物种。
传染性皮下及造血组织坏死病毒IHHNV于1981年首次在美国夏威夷地区的细角滨对虾中发现,随后在美洲和亚洲多个国家发现。宿主种类多如:细角滨对虾、凡纳滨对虾、罗氏沼虾等,对幼虾的的危害较大。虽然IHHNV不引起南美白对虾的死亡,但是会造成南美白对虾头胸甲、触角等畸形并且生长缓慢,其临床症状被称为慢性矮小残缺综合症。
虾肝肠胞虫EHP是2009年发现的微孢子虫新种,严格细胞内寄生,亚太地区多数国家的养殖对虾中均有发现。对虾感染该寄生虫后,会导致对虾出现生长缓慢或生长停滞,有时会出现白便等症状,但其不影响对虾的摄食和存活率。目前认为EHP是影响对虾生长缓慢的最主要的病原,成为对虾养殖的最大威胁之一,严重影响了对虾养殖业的产量增长,造成了严重的经济损失。这种疾病在对虾的仔虾期直至养成期均有感染的迹象,并且该疾病有大规模爆发的趋势
对虾肝胰腺坏死病NHP是由一种类立克次体的细菌肝胰腺坏死性细菌(Necrotizing hepato pancreatitis bacterium,NHPB)引起的对虾疾病。NHPB是一种革兰氏阴性菌,多形态,专性细胞内寄生。NHPB是一种感染能力很强的菌种,宿主包括南美白对虾、白滨对虾、细角滨对虾等。病原的传播方式主要是水平传播—通过污染的水体、食物等。
目前,对虾病害种类较多,诊断手段主要包括病理学和生物学方法、免疫诊断法、分子杂交及PCR技术。已建立的常规PCR技术,常是一次只能检测一种病原,比较费时间。而多重PCR技术是一种特殊的PCR方法,在一个反应体系中加入多对病原特异性引物,能够同时检测多种病原体,从而缩短病原检测的周期,提高检测效率。但多重PCR检测也受多种因素的影响,比如DNA聚合酶的活性、模板的质量、所设计的病原引物的特异性、PCR反应条件、不同病原样本间对试剂的竞争等因素都会影响检测结果的敏感性和准确性。
基于当前现状,需建立一种准确、快速、简便的对虾病原检测方法,以利于对病原的早期诊断,便于及时采取有效的防治措施。
发明内容
针对现有技术中的上述不足,本发明提供一种同时检测WSSV、IHHNV、EHP和NHP的多重PCR引物探针组合及方法和用途,本发明目的在于提供一种方便快捷,检测限低,且特异性强、灵敏度高、准确率高、且能同时检测对虾白斑综合征病毒、对虾传染性皮下及造血组织坏死病毒、虾肝肠胞虫、对虾肝胰腺坏死病等四种病原菌的多重荧光PCR检测方法。
为实现上述目的,本发明解决其技术问题所采用的技术方案是:
一种同时检测WSSV、IHHNV、EHP和NHP的多重PCR引物探针组合,该多重PCR引物组合所包括的引物和探针序列如下:
WSSV-F:5’-TGGTCCCGTCCTCATCTCAG-3’;(SEQ ID NO.1)
WSSV-R:5’-GCTGCCTTGCCGGAAATTA-3’;(SEQ ID NO.2)
WSSV-探针:5’-AGCCATGAAGAATGCCGTCTATCACACA-3’;(SEQ ID NO.3)
IHHNV-F:5’-TACTCCGGACACCCAACCA-3’;(SEQ ID NO.4)
IHHNV-R:5’-GGCTCTGGCAGCAAAGGTAA-3’;(SEQ ID NO.5)
IHHNV-探针:5’-ACCAGACATAGAGCTACAATCCTCGCCTATTTG-3’;(SEQ ID NO.6)
EHP-F:5’-AGTAAACTATGCCGACAA-3’;(SEQ ID NO.7)
EHP-R:5’-AATTAAGCAGCACAATCC-3’;(SEQ ID NO.8)
EHP-探针:5’-TCCTGGTAGTGTCCTTCCGT-3’;(SEQ ID NO.9)
NHP-F:5’-CGTTCACGGGCCTTGTACAC-3’;(SEQ ID NO.10)
NHP-R:5’-GCTCATCGCCTTAAAGAAAAGATAA-3’;(SEQ ID NO.11)
NHP-探针:5’-CCGCCCGTCAAGCCATGGAA-3’。(SEQ ID NO.12)
本发明用于检测WSSV、IHHNV、EHP和NHP的引物组包括:WSSV的VP664基因、IHHNV的非结构蛋白2(non-structural protein 2)基因、EHP的小亚基核糖体RNA(small subunitribosomal RNA)基因、NHP的16SrRNA基因所对应的特异性引物探针序列组;引物和探针的特异性强、灵敏度高,可应用荧光PCR方法同时检测WSSV、IHHNV、EHP和NHP。
进一步地,在探针的3’端连接有淬灭基团,并在不同探针的5’端连接有不同的荧光基团。
进一步地,淬灭基团为BHQ1或BHQ2;所述荧光基团为CY5、VIC、Texas Red或6-FAM。
一种试剂盒,包括上述多重PCR引物探针组合。
本发明用于检测WSSV、IHHNV、EHP和NHP的多重荧光PCR试剂盒包括PCR Mix、Buffer、阴性对照和阳性对照,PCR Mix含有WSSV、IHHNV、EHP和NHP对应的特异性引物探针。
一种多重PCR芯片,包括上述多重PCR引物探针组合。
上述引物探针组合、试剂盒,或多重PCR芯片在检测鉴定对虾WSSV、IHHNV、EHP和NHP四种病毒中的用途。
一种对虾WSSV、IHHNV、EHP和NHP四种病原菌进行检测的多重荧光PCR方法,包括以下步骤:
(1)以待测样本的DNA或cDNA为模板,使用权利要求1~3任一项所述的多重PCR引物探针组合或权利要求4所述的试剂盒进行PCR扩增;
(2)根据有无扩增曲线和Ct值,判断待测样本是否含有WSSV、IHHNV、EHP和NHP四种病原菌。
进一步地,若有扩增曲线,且满足Ct≤35,说明待测样本为所述多重荧光PCR引物探针组的荧光基团对应的WSSV、IHHNV、EHP和NHP相关病原菌;若无扩增曲线,说明待测样本不是四种病原菌中的任何一种。
进一步地,PCR扩增的扩增体系包括以下组分:2×TaqMan Fast qPCR Master Mix10μL,上下游引物各0.4μL,探针各0.2μL。模板DNA1μL,ddH2O补足至20μL。
进一步地,PCR扩增的扩增程序为:95℃预变性5min;95℃变性10s,62℃检测信号30s,循环40次。
本发明的有益效果:
本发明提供的基于该组引物和试剂盒的检测方法具有操作简单、灵敏度高、特异性强、检测速度快的优点,能够同时检测对虾WSSV、IHHNV、EHP和NHP这四种病原菌,大大节约检测时间。
附图说明
图1为本发明实施例1提供的阳性对照品和阴性对照品扩增结果图;其中1-4为阳性对照品扩增结果:1是WSSV质粒模板,2是IHHNV质粒模板,3是EHP质粒模板,4是NHP质粒模板,5为阴性对照品扩增结果;
图2为本发明实施例2提供的多重荧光定量PCR体系中WSSV检测的灵敏度实验结果图;从左到右依次为1×106copies/μL-1×10-1copies/μL的标准质粒扩增结果;
图3为本发明实施例2提供的多重荧光定量PCR体系中IHHNV检测的灵敏度实验结果图;从左到右依次为1×106copies/μL-1×10-1copies/μL的标准质粒扩增结果;
图4为本发明实施例2提供的多重荧光定量PCR体系中EHP检测的灵敏度实验结果图;从左到右依次为1×106copies/μL-1×10-1copies/μL的标准质粒扩增结果;
图5为本发明实施例2提供的多重荧光定量PCR体系中NHP检测的灵敏度实验结果图;从左到右依次为1×106copies/μL-1×10-1copies/μL的标准质粒扩增结果;
图6为多重荧光定量PCR的标准曲线图;其中WSSV的相关系数R2=0.997,IHHNV相关系数R2=0.999,EHP相关系数R2=0.999;NHP相关系数R2=0.998。
具体实施方式
下面对本发明的具体实施方式进行描述,以便于本技术领域的技术人员理解本发明,但应该清楚,本发明不限于具体实施方式的范围,对本技术领域的普通技术人员来讲,只要各种变化在所附的权利要求限定和确定的本发明的精神和范围内,这些变化是显而易见的,一切利用本发明构思的发明创造均在保护之列。
实施例1同时检测对虾WSSV、IHHNV、EHP和NHP四种病原菌的荧光定量PCR方法
1、取对虾样品,用细菌DNA提取试剂盒(DC103,诺唯赞,南京)/病毒RNA提取试剂盒(R401,诺唯赞,南京)进行核酸提取,得到样本中的总核酸,即总DNA和总RNA,于-20℃保存;
2、取一部分步骤1中提取的总核酸,运用RNA逆转录试剂盒(R312,诺唯赞,南京)进行反转录,得到对应样本cDNA;混合待测样本的DNA和cDNA,得到待检样本核酸。
3、目标基因的选择和引物探针的设计,见表1:
表1上下游引物和探针的序列信息
4、打开qPCR仪器预热准备检测,取出qPCR仪器对应的反应试剂八连管,进行样本核酸、阳性对照品和阴性对照品的加样,每个样孔体系均为20μL;样本核酸孔:以样本核酸为模板,分别加入10μL的2×TaqMan Fast qPCR Master Mix酶混合液,WSSV、IHHNV、EHP、NHP的上游和下游引物各0.4μL,WSSV、IHHNV、EHP、NHP的探针各0.2μL,样本核酸模板1μL,ddH2O补足至20μL;阳性对照孔:分别加入10μL的2×TaqMan Fast qPCR Master Mix酶混合液,WSSV、IHHNV、EHP、NHP的上游和下游引物各0.4μL,WSSV、IHHNV、EHP、NHP的探针各0.2μL,1μL阳性对照品(包含WSSV、IHHNV、EHP、NHP的质粒,拷贝数均为1×108copies/μL)模板1μL,ddH2O补足至20μL;阴性对照孔:分别加入10μL的2×TaqMan Fast qPCR Master Mix酶混合液,WSSV、IHHNV、EHP、NHP的上游和下游引物各0.4μL,WSSV、IHHNV、EHP、NHP的探针各0.2μL,ddH2O补足至20μL。
5、将加好样的八联管上机检测。循环条件设置:95℃预变性5min;95℃变性10s,62℃检测信号30s,循环40次。仪器检测通道选择:荧光通道设定为CY5、VIC、Texas Red、6-FAM。
6、收集仪器反应结束每个样孔的ct值,进行有效性判断。阴性对照品应无Ct值或者为0,阳性对照品Ct值应≤35,否则,本次实验结果无效。阳性对照品和阴性对照品的扩增结果见图1,其中1-4为阳性对照品扩增结果:1是WSSV质粒模板,2是IHHNV质粒模板,3是EHP质粒模板,4是NHP质粒模板,5为阴性对照品扩增结果;
7、确定实验结果有效后,对样本阳性进行判断。CY5通道Ct值≤35时视为WSSV阳性,否则为WSSV阴性;VIC通道Ct值≤35时视为IHHNV阳性,否则为IHHNV阴性;Texas Red通道Ct值≤35时视为EHP阳性,否则为EHP阴性;6-FAM通道Ct值≤35时视为NHP阳性,否则为NHP阴性;
实施例2同时检测对虾WSSV、IHHNV、EHP和NHP四种病原菌的荧光定量PCR灵敏性分析
1、取阳性对照品,里面含有对虾WSSV、IHHNV、EHP和NHP四种病原菌的重组质粒模板,所有模板拷贝数均为1×107copies/μL。对阳性对照品进行10倍梯度稀释,稀释8个梯度为1×106copies/μL-1×10-1copies/μL。
2、将阳性对照品和步骤1中所稀释的8个梯度质粒样组成一套10倍梯度稀释的标准质粒样,进行加样。打开qPCR仪器预热准备检测,取出qPCR仪器对应的反应试剂八连管,进行标准质粒样孔和阴性对照品孔的加样,每个样孔体系均为20μL。标准质粒样孔:以稀释好的每个质粒样为模板,分别加入分别加入10μL的2×TaqMan Fast qPCR Master Mix酶混合液,WSSV、IHHNV、EHP、NHP的上游和下游引物各0.4μL,WSSV、IHHNV、EHP、NHP的探针各0.2μL,样本核酸模板1μL,ddH2O补足至20μL;阴性对照孔:分别加入10μL的2×TaqMan FastqPCR Master Mix酶混合液,WSSV、IHHNV、EHP、NHP的上游和下游引物各0.4μL,WSSV、IHHNV、EHP、NHP的探针各0.2μL,ddH2O补足至20μL;
3、将加好样的八联管上机检测。循环条件设置:95℃预变性5min;95℃变性10s,62℃检测信号30s,循环40次。仪器检测通道选择:荧光通道设定为CY5、VIC、Texas Red、6-FAM。
4、收集仪器反应结束每个样孔的ct值,进行有效性判断。阴性对照品应无Ct值或者为0,阳性对照品Ct值应≤35,否则,本次实验结果无效。
5、确定实验结果有效后,进行标准曲线的构建,得出反应最低检测限。
图2为多重荧光定量PCR体系中WSSV检测的灵敏度实验结果图,从图中可以看出,1×106copies/μL-1×100copies/μL都有扩增,说明对WSSV的检测灵敏度为1×100copies/μL。
图3为多重荧光定量PCR体系中IHHNV检测的灵敏度实验结果图,从图中可以看出,1×106copies/μL-1×100copies/μL都有扩增,说明对IHHNV的检测灵敏度为1×100copies/μL。
图4为多重荧光定量PCR体系中EHP检测的灵敏度实验结果图,从图中可以看出,1×106copies/μL-1×100copies/μL都有扩增,说明对EHP的检测灵敏度为1×100copies/μL。
图5为多重荧光定量PCR体系中NHP检测的灵敏度实验结果图,从图中可以看出,1×106copies/μL-1×100copies/μL都有扩增,说明对NHP的检测灵敏度为1×100copies/μL。
图6为多重荧光定量PCR的标准曲线图;根据WSSV、IHHNV、EHP、NHP的标准质粒扩增结果构建标准曲线图,其中WSSV相关系数R2=0.997;IHHNV相关系数R2=0.999;EHP相关系数R2=0.999;NHP相关系数R2=0.998。
结合图2-6实验结果可知,所有病毒相关系数均大于0.995,本发明对对虾四种病原菌WSSV、IHHNV、EHP、NHP的最低检出限都达到了1×100copies/μL。
实施例3对虾WSSV、IHHNV、EHP和NHP四种病原菌的质粒构建
本试剂盒中的阳性对照品内含有WSSV、IHHNV、EHP和NHP四种病原菌的重组质粒模板,所有模板拷贝数均为1×108copies/μL。质粒构建过程如下:
人工合成目的片段的基因,将其连接在pMD18-T质粒上,分别得到连接有WSSV、IHHNV、EHP和NHP四种病原菌的重组质粒。
WSSV的人工合成目的片段序列为:
AACGAACTGGCCGGCACCATCGCTGAATCTGTACCAGAAAGCGTATATGAAAACACCAAGGAAATGATTGATAGACTAGGCTCTGACGACCTCTTCAAATCTAATAATAATGGAGGAGTAGAATCAATGGATTATGAAGATAGCGAAACAACATCCAACAATGGTCCCGTCCTCATCTCAGAAGCCATGAAGAATGCCGTCTATCACACACTAATTTCCGGCAAGGCAGCTCGCCCGGAAAATGTACCATT CGCCTCATGCGCCAGCGGCCCTCTCGCCTTTGATTTCCTTCTGTCAAA。
IHHNV的人工合成目的片段序列为:
TTCACCATTACAGATCATGGTGACCACTGGCACATCACATACTCCGGACACCCAACCAATAAGACCAGACATAGAGCTACAATCCTCGCCTATTTGGGAGTTACCTTTGCTGCCAGAGCCGAAGCTGAAGCGACTACGGTACTTGTTAGAAATATCAAGAGATGGATACTCTATCTTATCAGATACGGTATTGAACGGCTTTCGTATTTTGGTCTTGGCCACGCCATTTTTAAACGAATCATCAAATACTTCCAACAATACAGAAGAGACGAAGACGCAGTAGACGGACCATGTCCATA。
EHP的人工合成目的片段序列为:
CTGGGGATCAAGGACGAAGGCTAGAGTATCGAAAGTGATTAGACACCGCTGTAGTTCTAGCAGTAAACTATGCCGACAATGCTGGGTGTTGCGAGAGCGATGCTTGGTGTGGGAGAAATCTTAGTTTTCGGGCTCTGGGGATAGTACGCTCGCAAGGGTGAAACTTAAAGCGAAATTGACGGAAGGACACTACCAGGAGTGGATTGTGCTGCTTAATTTAACTCAACGCGGGAAAACTTACCAGGGTCAAGTCTATCGTAGATTGGAGACATGAGGTAGACAAGAGTGGTGCATGGCCG。
NHP的人工合成目的片段序列为:
TCAGGTCCTCATGGCCCTTATGGGCTGGGCTACACACGTGCTACAATGGCGATCACAATGAGAAGCAAGGGGGTGACCCGGAGCCAATCTCTAAAAATCGTCTCAGTTCGGATTGTTCTCTGCAACTCGAGAGCATGAAGTTGGAATCGCTAGTAATCGCAGATCAGCATGCTGCGGTGAATACGTTCACGGGCCTTGTACACACCGCCCGTCAAGCCATGGAAGTTATCTTTTCTTTAAGGCGATGAGCTAACCAGCAATGGAGGCAGTCGACCACAGAAAAGGTGATGACTGGGGTT。
最后应说明的是,以上具体实施方式仅用以说明本发明的技术方案而非限制,尽管参照实例对本发明进行了详细说明,本领域的普通技术人员应当理解,可以对本发明的技术方案进行修改或者等同替换,而不脱离本发明技术方案的精神和范围,其均应涵盖在本发明的权利要求范围当中。
Claims (10)
1.一种同时检测WSSV、IHHNV、EHP和NHP的多重PCR引物探针组合,其特征在于,所述多重PCR引物组合所包括的引物序列如下:
WSSV-F:5’-TGGTCCCGTCCTCATCTCAG-3’;
WSSV-R:5’-GCTGCCTTGCCGGAAATTA-3’;
WSSV-探针:5’-AGCCATGAAGAATGCCGTCTATCACACA-3’;
IHHNV-F:5’-TACTCCGGACACCCAACCA-3’;
IHHNV-R:5’-GGCTCTGGCAGCAAAGGTAA-3’;
IHHNV-探针:5’-ACCAGACATAGAGCTACAATCCTCGCCTATTTG-3’;
EHP-F:5’-AGTAAACTATGCCGACAA-3’;
EHP-R:5’-AATTAAGCAGCACAATCC-3’;
EHP-探针:5’-TCCTGGTAGTGTCCTTCCGT-3’;
NHP-F:5’-CGTTCACGGGCCTTGTACAC-3’;
NHP-R:5’-GCTCATCGCCTTAAAGAAAAGATAA-3’;
NHP-探针:5’-CCGCCCGTCAAGCCATGGAA-3’。
2.根据权利要求1所述的多重PCR引物探针组合,其特征在于,在探针的3’端连接有淬灭基团,并在不同探针的5’端连接有不同的荧光基团。
3.根据权利要求1所述的多重PCR引物探针组合,其特征在于,所述淬灭基团为BHQ1或BHQ2;所述荧光基团为CY5、VIC、Texas Red或6-FAM。
4.一种试剂盒,其特征在于,包括权利要求1~3任一项所述的多重PCR引物探针组合。
5.一种多重PCR芯片,其特征在于,包括权利要求1~3任一项所述的多重PCR引物探针组合。
6.权利要求1~3所述的引物探针组合、权利要求4所述的试剂盒,或权利要求5所述的多重PCR芯片在检测鉴定对虾WSSV、IHHNV、EHP和NHP四种病毒中的用途。
7.一种对虾WSSV、IHHNV、EHP和NHP四种病原菌进行检测的多重荧光PCR方法,其特征在于,包括以下步骤:
(1)以待测样本的DNA或cDNA为模板,使用权利要求1~3任一项所述的多重PCR引物探针组合或权利要求4所述的试剂盒进行PCR扩增;
(2)根据有无扩增曲线和Ct值,判断待测样本是否含有WSSV、IHHNV、EHP和NHP四种病原菌。
8.根据权利要求7所述的多重荧光PCR方法,其特征在于,若有扩增曲线,且满足Ct≤35,说明待测样本为所述多重荧光PCR引物探针组的荧光基团对应的WSSV、IHHNV、EHP和NHP相关病原菌;若无扩增曲线,说明待测样本不是四种病原菌中的任何一种。
9.根据权利要求7所述的多重荧光PCR方法,其特征在于,所述PCR扩增的扩增体系包括以下组分:2×TaqMan Fast qPCR Master Mix 10μL,上下游引物各0.4μL,探针各0.2μL。模板DNA1μL,ddH2O补足至20μL。
10.根据权利要求7所诉的多重荧光PCR方法,其特征在于,所述的PCR扩增的扩增程序为:95℃预变性5min;95℃变性10s,62℃检测信号30s,循环40次。
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