CN115612753A - 一种阿留申、肠炎、犬瘟热病检测的方法 - Google Patents
一种阿留申、肠炎、犬瘟热病检测的方法 Download PDFInfo
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Abstract
本发明涉及生物检疫领域，具体涉及一种用于水貂阿留申病毒、肠炎病毒、犬瘟热病毒的三重TaqMan荧光定量PCR的引物和探针及其应用。该方法仅特异性扩增CDV、AMDV和MEV，与水貂的其他病原无交叉反应，特异性强；对AMDV与MEV、CDV的质粒标准品的最低检测限为7.5×101copies/μL，具有较高的敏感性；组内和组间的变异系数均小于4％，具有良好的重复性。
Description
技术领域
本发明属于生物检疫领域，具体涉及一种三重TaqMan荧光定量RT-PCR检测方法。
背景技术
犬瘟热病毒(Canine distemper virus，CDV)是一种急性、高度接触性传染病，主要侵害易感动物的消化系统、神经系统和呼吸系统，致使水貂发病后，死亡率可高达30-80％，在雪貂上高达100％。CDV感染宿主范围广，目前已从犬科动物和鼬科动物、浣熊科、食肉目所有8个科及偶蹄目猪科、灵长目猕猴属等多种动物体内分离到该病毒，近年来我国多地也相继暴发该病毒病，临床表现为出现明显的神经症状、体温升高、饮食欲下降，消化不良，口鼻部皮肤炎症、角化增厚结痂、排出腥臭味粪便，严重者衰竭而亡。尽管减毒疫苗在过去几年中大大降低了死亡率，并部分控制了该疾病，但在犬和野生动物宿主中仍报告了几次CDV的暴发。已成为严重危害养犬业、经济动物养殖业、野生动物保护事业的传染病之一。
水貂阿留申病毒(Mink Aleutian virus，AMDV)是一种细小病毒，可引起免疫复杂反应，导致水貂发生阿留申病。AMDV在其他种类的鼬科中也发现了自然感染，包括欧洲、日本和北美的雪貂、水貂(黄貂)、水貂、水獭。除了麝鼠科，还在浣熊、臭鼬、山猫、和狐狸中发现了AMDV感染的证据。AMDV可引起高丙种球蛋白血症、高抗AMDV抗体水平、感染性免疫复合物的形成和肾小球肾炎。死亡通常由肾功能衰竭引起。临床症状包括贫血、厌食症、凝血异常和不孕症。传播发生在水平和垂直传播，病毒存在于受感染动物的粪便、尿液和唾液中。由于该病的病原感染机制特殊，所以到目前为止，没有公认的免疫效果好的疫苗，也没有特效疗法，故对其防治极其困难。国内仅仅只能依靠检测阳性，淘汰净化来清除该病，但收效甚微。有国外学者从基因和蛋白水平对AD的研究较为深入，对其发病机理也日渐清晰。
水貂肠炎病毒(Mink enteritis virus，MEV)是细小病毒科细小病毒属的一员，是一种单链DNA病毒。MEV引起水貂病毒性肠炎，这是一种急性和高传染性的疾病，MEV主要感染肠系膜淋巴结和小肠上皮细胞，导致厌食、呕吐、严重腹泻和淋巴细胞减少，病毒主要通过粪便口服途径传播。MEV的诊断是控制该疾病的一个重要措施，尽管MEV现在有多种诊断方法，但是各种方法却存在着各自的缺点。例如，虽然电子显微镜和病毒分离具有高度的特异性和敏感性，但它们对于常规临床使用来说往往过于耗时和昂贵。此外，血液凝集试验(HA)快速但缺乏特异性，血凝抑制试验(HI)需要持续供应新鲜红细胞，不适合检测非血凝MEV分离株。近年来，尽管已有一种疫苗被用于预防该疾病的进一步传播，但感染的数量仍在继续增加。这可能与MEV持续突变的能力有关。
目前，国内外学者针对以上病原建立了RT-PCR、多重RT-PCR、荧光定量RT-PCR、ELISA等检测方法，但尚无同时区别检测CDV、MEV及AMDV的多重TaqMan荧光定量RT-PCR方法的报道。
发明内容
本研究针对CDV、MEV及AMDV特定基因，设计特异性引物和荧光探针，建立了同一体系中同时鉴别检测CDV、MEV及AMDV的多重TaqMan荧光定量RT-PCR方法，并应用于临床检测，为相应病原的检测和流行病学调查提供特异、高效、敏感的技术手段。具体技术方案如下：
(1)样品采集与制备
(2)病毒核酸提取
(3)取反应体系管置于室温融化，用移液器吸取模板2μL加入反应体系管中，用离心机离心30s，离心结束放入荧光定量PCR仪器中，使用程序进行扩增检测。
进一步的，样品采集过程具体操作为：将拭子用无菌生理盐水沾湿，收集眼鼻肛门分泌物样品，再将拭子插入无菌生理盐水采样管中，涡旋震荡混匀，取上清液200μL上清液于1.5ml PCR反应管中；粪便提取方法为取少量粪便，按1：10的比例加入生理盐水混匀后12000rpm离心，取200μL于1.5mlPCR反应管中。
进一步的，3.扩增程序为程序为37℃180s；95℃120s；95℃10s，52℃30s，40个循环。
所用探针与引物对的核苷酸序列为SEQ ID No.1，SEQ ID No.2，SEQ ID No.3，SEQID No.4，SEQ ID No.5，SEQ ID No.6，SEQ ID No.7，SEQ ID No.8，SEQ ID No.9.
本发明有益效果为：
该方法仅特异性扩增CDV、AMDV和MEV，与水貂的其他病原无交叉反应，特异性强；对AMDV与MEV、CDV的质粒标准品的最低检测限为7.5×101copies/μL，具有较高的敏感性；组内和组间的变异系数均小于4％，具有良好的重复性。利用该方法与已报道的AMDV、CDV、MEV多重PCR检测方法检测采集的200份腹泻临床样本，结果显示两者的符合率100％。本研究建立的方法为犬瘟热病毒(CDV)、水貂阿留申病毒(AMDV)、水貂肠炎病毒(MEV)的临床快速和特异性鉴别提供了一种切实可行的技术支撑。
说明书附图
图1多重TaqMan荧光定量RT-PCR标准曲线
图2多重TaqMan荧光定量RT-PCR特异性试验结果
图3多重TaqMan荧光定量RT-PCR的敏感性试验结果，
1～8：浓度分别为7.5×108～7.5×101拷贝/μL的pCDV-N；
9～16：浓度分别为7.5×108～7.5×101拷贝/μL的pMEV-VP2；
17～24：浓度分别为7.5×108～7.5×101拷贝/μL的pAMDV-NS1；25:阴性对照
1～8:The concentrations were 7.5×108-7.5×101copies/μL of pCDV-N,respectively.
9～16:The concentration was 7.5×108-7.5×101copies/μL of pMEV-VP2,respectively.
17～24:The concentrations were 7.5×108-7.5×101copies/μL of pAMDV-NS1,respectively；25:Negative control
具体实施例
临床样品的检测
对2022年山东省内各大养殖场收集的200份疑似患病的腹泻粪样，利用改良的核酸快速提取方法进行核酸提取，12000r/min瞬时离心后，取2μL按优化后的多重TaqMan荧光定量PCR检测。同时根据马芹建立的三重PCR同时检测，比较两者的检测结果，并计算两者的符合率。
1重组质粒标准品的鉴定结果
提取MEV、AMDV病毒液的总DNA，CDV的RNA反转录成cDNA后作为模板，利用特异性引物进行PCR扩增，获得MEV VP2基因(190bp)，CDV N基因(198bp)、AMDV NS1基因(119bp)目的片段，均与预期结果一致(图略)。构建的3种重组质粒分别均经PCR、酶切与测序鉴定均正确，表明正确构建了3种重组质粒pMEV-VP2、pCDV-N、pAMDV-NS1，经测定浓度，换算成拷贝数作为重组质粒标准品。
2多重TaqMan荧光定量RT-PCR最佳反应条件的确定
经过优化，多重TaqMan荧光定量RT-PCR的最佳反应体系为25μL：2×5G qPCRBuffer BB 12.5μL,RT-qPCR Enzyme Mix UD 1.3μL，CDV、MEV和AMDV的上游引物0.4μL,下游引物0.4μL，探针1μL,混合质粒模板2μL模板为重组质粒标准品pMEV-VP2、pCDV-N、pAMDV-NS1等体积混合物2μL，灭菌双蒸水补直25uL。扩增程序为：37℃180s；95℃120s；95℃10s，52℃30s，40个循环，同时收集荧光信号。
3多重TaqMan荧光定量RT-PCR标准曲线的建立
将重组质粒标准品pMEV-VP2、pCDV-N、pAMDV-NS1 10倍倍比稀释后等体积混合，取终浓度为7.5×107拷贝/μL～7.5×101拷贝/μL的质粒作为模板，利用本研究建立的方法进行扩增，获得多重TaqMan荧光定量RT-PCR扩增曲线及标准曲线(图1)。3种标准曲线的相关系数(R2)均在0.99及以上。表明，3种质粒标准品在浓度7.5×107拷贝/μL～7.5×101拷贝/μL与Ct值之间呈现良好的线性关系。
4特异性试验结果
分别以CCoV、CDV、CAV-2、CPIV的RNA及MEV、AMDV、CPV的DNA为模板,利用建立的多重TaqMan荧光定量RT-PCR进行扩增。结果显示，仅阳性对照的重组质粒标准品pMEV-VP2、pCDV-N、pAMDV-NS1及MEV、CDV、AMDV的核酸出现扩增曲线，且Ct值均小于35，而其他病毒的cDNA或DNA及阴性对照均无扩增曲线(图2)。表明，所建立的多重TaqMan荧光定量RT-PCR方法特异性较强。
5敏感性试验结果
以质粒标准品pMEV-VP2、pCDV-N、pAMDV-NS1 10倍倍比稀释后等体积混合物作为模板，进行多重TaqMan荧光定量RT-PCR的敏感性试验。结果显示，Ct值在35以下时，该方法对对pMEV-VP2、pAMDV-NS1、pCDV-N质粒标准品的检测下限为7.5×101拷贝/μL。(图3)表明，该方法敏感性较高。
6重复性试验结果
取3个不同浓度的重组质粒标准品(终浓度分别为7.5×107拷贝/μL、7.5×105拷贝/μL、7.5×103拷贝/μL)混合物，利用本研究建立的多重TaqMan荧光定量RT-PCR分别进行组内和组间的重复性试验。结果显示，组内与组间重复性试验Ct值的变异系数均小于4％(表1)。
表明，该方法重复性较好。
表1多重TaqMan荧光定量RT-PCR的重复性试验7临床检测
本研究建立的方法的检测结果显示，200份临床样本中，检出26份CDV，阳性率为13％；检出48份MEV，阳性率为24％；检出21份AMDV，阳性率为10.5％其中CDV和MEV混合感染15份，混合感染率为7.5％，CDV和AMDV混合感染8份，混合感染率4％；MEV和AMDV混合感染17份，混合感染率8.5％；三者共同感染8份，感染率4％。同时根据马芹建立的三重PCR同时检测。结果显示(表2)，该方法对这些样品的检测结果与本研究建立的方法检测结果一致，两者的符合率为100％。表明，本研究建立的方法可以用于临床样品检测。
表2多重TaqMan RT-qPCR检测方法与RT-PCR检测方法对临床样品检测结果的比较。
Claims (6)
1.一种水貂阿留申、肠炎、犬瘟热病毒三重TaqMan荧光定量RT-PCR检测方法，其步骤包括：
(1)样品采集与制备
(2)病毒核酸提取
(3)取反应体系管置于室温融化，用移液器吸取模板2μL加入反应体系管中，用离心机离心30s，离心结束放入荧光定量PCR仪器中，使用程序进行扩增检测。
2.根据权利要求1所述的水貂阿留申、肠炎、犬瘟热病毒三重TaqMan荧光定量RT-PCR检测方法，其特征在于，样品采集过程具体操作为：将拭子用无菌生理盐水沾湿，收集眼鼻肛门分泌物样品，再将拭子插入无菌生理盐水采样管中，涡旋震荡混匀，取上清液200μL于1.5ml PCR反应管中；粪便提取方法为取少量粪便，按1：10的比例加入生理盐水混匀后12000rpm离心，取200μL上清液于1.5ml PCR反应管中。
3.根据权利要求1所述的水貂阿留申、肠炎、犬瘟热病毒三重TaqMan荧光定量RT-PCR检测方法，其特征在于，扩增程序为程序为37℃180s；95℃120s；95℃10s，52℃30s，40个循环。
4.一组用于水貂阿留申、肠炎、犬瘟热病毒三重TaqMan荧光定量RT-PCR检测的探针和引物，其特征在于，所述探针与引物的核苷酸序列为SEQ ID No.1，SEQ ID No.2，SEQ IDNo.3，SEQ ID No.4，SEQ ID No.5，SEQ ID No.6，SEQ ID No.7，SEQ ID No.8，SEQ ID No.9.
5.如权利要求4所述探针与引物在水貂阿留申、肠炎、犬瘟热病毒检测中的应用。
6.如权利要求1所述的三重TaqMan荧光定量RT-PCR检测方法在水貂阿留申、肠炎、犬瘟热病毒检测中的应用。
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| 马芹;王元智;闫文卓;赵丽丽;陈洪岩;陆涛峰;: "水貂阿留申病毒、肠炎病毒与犬瘟热病毒多重PCR检测方法的建立与应用", 中国比较医学杂志, no. 12, 30 December 2017 (2017-12-30), pages 97 - 101 * |
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