CN107164559A - 基于POCKIT Micro荧光PCR平台的禽流感病毒H7亚型检测试剂及应用 - Google Patents
基于POCKIT Micro荧光PCR平台的禽流感病毒H7亚型检测试剂及应用 Download PDFInfo
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- CN107164559A CN107164559A CN201710366849.4A CN201710366849A CN107164559A CN 107164559 A CN107164559 A CN 107164559A CN 201710366849 A CN201710366849 A CN 201710366849A CN 107164559 A CN107164559 A CN 107164559A
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
- C12Q1/701—Specific hybridization probes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
Abstract
本发明属于生物技术领域，尤其涉及一种基于POCKIT Micro荧光PCR平台的禽流感病毒H7亚型检测试剂及应用。本发明检测试剂中包含检测禽流感病毒H7亚型的特异性引物和探针；检测试剂盒中包含标准品和荧光定量PCR反应试剂，荧光定量PCR反应试剂包含dNTPs、Taq酶、缓冲液、特异性引物和探针；检测系统中包含核酸萃取试剂盒、检测试剂盒、POCKIT Micro荧光PCR核酸分析仪、微型离心机和移动电源。本发明检测试剂盒和检测系统具有检测结果准确、操作简单、方便易携、节省试剂、无需电泳、现场快速检测等优点，可广泛应用于禽流感的早期筛查和诊断。
Description
基于POCK IT M i cro荧光PCR平台的禽流感病毒H7亚型检测试 剂及应用
技术领域
[0001] 本发明属于生物技术领域，尤其涉及一种基于POCKIT Micro荧光PCR平台的禽流 感病毒H7亚型检测试剂及应用，本发明还涉及针对禽流感病毒H7亚型的检测试剂盒及检测 系统。
背景技术
[0002] 禽流感(Avian Influenza)是禽流行性感冒的简称，它是由禽流感病毒引起的一 种禽类急性传染病，被国际兽疫局定为甲类传染病。通常禽流感只在禽类之间感染和传播， 但有时也能感染人类，自1997年香港首次发现人感染禽流感以来，全球多地均报告了人禽 流感病例，病死率约60%。虽然禽流感被发现已逾百年，但至今人类并未掌握特异性的预防 和治疗方法，仅能以消毒、隔离、大量宰杀禽畜的方法防止其蔓延。因此，禽流感目前已成为 一个重大的公共安全问题，在给禽类和人类健康带来严重威胁的同时，也给社会造成了巨 大的经济损失。
[0003] 禽流感病毒(AIV)属于正粘病毒科的甲型流感病毒，多为球形，直径80〜120nm，有 囊膜，病毒表面有10〜12nm的密集钉状物或纤突覆盖，病毒囊膜内有螺旋形核衣壳。禽流感 病毒基因组由8个负链的单链RNA片段组成，共编码10个病毒蛋白。依据其套膜上的血凝素 (H) /和神经氨酸酶(N)蛋白抗原性的不同，可分为16个H亚型（Hl〜H16)和9个N亚型（NI〜 N9)。在禽类中高致病性禽流感病毒为H5、H7亚型，典型的鸡禽流感病毒为H7N7型，人感染禽 流感病毒主要为H5N1、H9N2、H7N7型。
[0004] H7亚型是1955年被发现并确定的禽流感病毒亚型，作为一种高致病性禽流感病 毒，近年来，H7亚型病毒的暴发已导致全球超过七千万家禽的死亡，疫情波及欧洲、亚洲、北 美洲、南美洲和大洋洲，由于该亚型病毒多为暴发型且破坏力巨大，因此引发了全球的广泛 关注。虽然目前针对H7亚型的基础研究已取得了一定的进展，例如已经测定获得了其HA基 因的全部序列以及血凝素基因的部分序列，但我们的研究还有很大的局限，对病毒进化、致 病机制、流行规律等仍不甚了解，因此，只有进一步加强在人群和动物中对该亚型禽流感病 毒的监测，同时致力于基础研究和相关疫苗及药物的研发，才能使我们从容地应对未来可 能出现的禽流感疫情。为了实现上述目的，研究开发针对禽流感病毒H7亚型的检测试剂，并 建立操作简便、快速、敏感性好、特异性强的检测方法和检测系统是很有必要的，将其推广 应用于禽流感的早期筛查会对疫情的预防和控制产生积极的影响。
发明内容
[0005] 针对上述问题，本发明人研究开发了一种基于POCKIT Micro荧光PCR平台的禽流 感病毒H7亚型检测试剂，并在该检测试剂的基础上建立了禽流感病毒H7亚型的检测试剂盒 及检测系统。本发明检测试剂盒及检测系统操作简单、检测速度快、敏感性好、特异性强、方 便易携带且检测成本较低。
[0006] 利用本发明检测试剂盒及检测系统可在实验室、医院、畜牧场、饲养场、活禽市场 等各种场所即时的对禽流感病毒H7亚型进行检测，并能够准确、快速的获得检测结果。
[0007] 第一方面，本发明提供了一种基于POCKIT Micro荧光PCR平台的禽流感病毒H7亚 型检测试剂，所述检测试剂中包含检测禽流感病毒H7亚型的特异性引物和探针，其中，所述 特异性引物的核苷酸序列如SEQ ID NO: 1和SEQ ID N0:2所示，所述探针的核苷酸序列如 SEQ ID NO:3和SEQ ID NO:4所示，所述探针的核苷酸序列的5’端连接有荧光报告基团FAM 标记，3 ’端连接有非荧光淬灭基团和MGB修饰基团。
[0008] 进一步地，本发明涉及上述检测试剂在制备禽流感病毒H7亚型检测试剂盒中的应 用。
[0009] 尤其是，本发明还涉及上述检测试剂在制备禽流感病毒H7N7或H7N9亚型检测试剂 盒中的应用。
[00Ί0] 此外，本发明还涉及上述检测试剂在制备H7亚型禽流感诊断或治疗药物中的应 用。
[0011] 第二方面，本发明提供了一种基于POCKIT Micro荧光PCR平台的禽流感病毒H7亚 型检测试剂盒，所述检测试剂盒中包含标准品和荧光定量PCR反应试剂;所述荧光定量PCR 反应试剂包含dNTPs、Taq酶、缓冲液、特异性引物和探针;其中，所述特异性引物的核苷酸序 列如SEQ ID NO: 1和SEQ ID N0:2所示，所述探针的核苷酸序列如SEQ ID N0:3和SEQ ID NO: 4所示，所述探针的核苷酸序列的5 ’端连接有荧光报告基团FAM标记，3 ’端连接有非荧光 淬灭基团和MGB修饰基团。
[0012] 进一步地，上述检测试剂盒中还包含阳性对照品和/或阴性对照品。
[0013] 优选地，上述检测试剂盒中的标准品为包含禽流感病毒H7亚型HA基因或该基因片 段的重组质粒。
[0014] 优选地，上述检测试剂盒中的荧光定量PCR反应试剂为冷冻干燥粉，该检测试剂盒 中还包含CldH2O。
[0015] 利用冷冻干燥技术将荧光定量PCR反应试剂制成冷冻干燥粉，可有效延长其保质 期，所述荧光定量PCR反应试剂冷冻干燥粉在4°C条件下的保质期可达2年，且短途运输过程 中无需冷链。
[0016] 第三方面，本发明提供了一种基于POCKIT Micro荧光PCR平台的禽流感病毒H7亚 型检测系统，所述检测系统中包含核酸萃取试剂盒、检测试剂盒、POCKIT Micro荧光PCR核 酸分析仪、微型离心机和移动电源；
[0017] 所述检测试剂盒中包含标准品和荧光定量PCR反应试剂;所述荧光定量PCR反应试 剂包含dNTPs、Taq酶、缓冲液、特异性引物和探针；其中，所述特异性引物的核苷酸序列如 SEQ ID N0:1和SEQ ID N0:2所示，所述探针的核苷酸序列如SEQ ID N0:3和SEQ ID N0:4所 示，所述探针的核苷酸序列的5 ’端连接有荧光报告基团FAM标记，3 ’端连接有非荧光淬灭基 团和MGB修饰基团。
[0018] 优选地，上述检测系统，其中所述检测试剂盒中包含的标准品为包含禽流感病毒 H7亚型HA基因或该基因片段的重组质粒;所述荧光定量PCR反应试剂为冷冻干燥粉，所述检 测试剂盒中还包含CldH2O。
[0019] 本发明检测试剂盒和检测系统由于具有检测结果准确、操作简单、方便易携、节省 试剂、无需电泳、现场快速检测等优点，可将其推广应用于禽流感的早期筛查和诊断，必定 会对禽流感疫情的预防和控制产生积极的影响。
具体实施方式
[0020] 以下通过特定的具体实例说明本发明的实施方式，本领域技术人员可由本说明书 所揭露的内容轻易地了解本发明的其他优点与功效。本发明还可以通过另外不同的具体实 施方式加以实施或应用，本说明书中的各项细节也可以基于不同观点与应用，在没有背离 本发明的精神下进行各种修饰或改变。
[0021] 在进一步描述本发明具体实施方式之前，应理解，本发明的保护范围不局限于下 述特定的具体实施方案;还应当理解，本发明实施例中使用的术语是为了描述特定的具体 实施方案，而不是为了限制本发明的保护范围。
[0022] 除非另外定义，本发明中使用的所有技术和科学术语与本技术领域技术人员通常 理解的意义相同。除实施例中使用的具体方法、设备、材料外，根据本技术领域的技术人员 对现有技术的掌握及本发明的记载，还可以使用与本发明实施例中所述的方法、设备、材料 相似或等同的现有技术的任何方法、设备和材料来实现本发明。
[0023] 下列实施例中未注明具体条件的实验方法，通常按照常规条件如J. Sambrook等 著，分子克隆：实验室手册（New York:Cold Spring Harbor Laboratory Press, 1989)中所 述的条件，或按照实验器材制造厂商所建议的条件。
[0024] 实施例1禽流感病毒H7亚型检测试剂盒1
[0025] 本检测试剂盒中包含标准品和荧光定量PCR反应试剂。
[0026] 其中，标准品为包含禽流感病毒H7亚型HA基因或该基因片段的重组质粒。
[0027] 荧光定量PCR反应试剂包含dNTPs、Taq酶、缓冲液、特异性引物和探针，特异性引物 和探针的核苷酸序列如下表1所示。
[0028] 表1特异性引物和探针的核苷酸序列
[0029]
[0030] 实施例2禽流感病毒H7亚型检测试剂盒2
[0031] 本检测试剂盒中包含标准品、荧光定量PCR反应试剂冷冻干燥粉、ddH20、阳性对照 品和阴性对照品。
[0032] 其中，标准品为包含禽流感病毒H7亚型HA基因或该基因片段的重组质粒。
[0033] 荧光定量PCR反应试剂冷冻干燥粉包含dNTPs、Taq酶、缓冲盐、特异性引物和探针， 特异性引物和探针的核苷酸序列如上表1所示。
[0034] 阳性对照品为含有禽流感病毒H7亚型HA基因或该基因片段的DNA样品，阴性对照 品为不含有HA基因或该基因片段的DNA样品。
[0035] 实施例3禽流感病毒H7亚型检测系统
[0036] 本检测系统中包含核酸萃取试剂盒、检测试剂盒、POCKIT Micro荧光PCR核酸分析 仪、微型离心机和移动电源。
[0037] 检测试剂盒中包含标准品和荧光定量PCR反应试剂。
[0038] 其中，标准品为包含禽流感病毒H7亚型HA基因或该基因片段的重组质粒。
[0039] 荧光定量PCR反应试剂包含dNTPs、Taq酶、缓冲液、特异性引物和探针，特异性引物 和探针的核苷酸序列如上表1所示。
[0040] 实施例4禽流感病毒H7亚型检测
[0041] —、检测对象
[0042] 经实验室PCR方法确诊的感染H7亚型禽流感病毒的家禽咽喉拭子为检测样本，未 感染H7亚型禽流感病毒的家禽咽喉拭子作为阴性对照样本。
[0043] 二、样品核酸提取
[0044] 实验试剂：
[0045] 使用PetNAD核酸萃取试剂盒(金瑞鸿捷生物科技有限公司），应用纯化管柱萃取系 统。
[0046] 萃取试剂盒产品组成如下表2所示。
[0047] 表2萃取试剂盒产品组成
[0048]
[0049] 实验仪器：
[0050] Cubee微型离心机(瑞基海洋生物科技股份有限公司）。
[0051 ] 提取方法：
[0052] 按照PetNAD核酸萃取试剂盒使用说明书，提取样品基因组DNA，操作步骤如下：
[0053] 1、样本的处理
[0054] 分别将检测样本和阴性对照样本的咽喉拭子放置在含有Iml DEPC水或是生理盐 水中充分震荡混合，丢弃拭子，置于微型离心机中先离心数秒，取200μ1上清液备用。
[0055] 2、核酸提取
[0056] (1)取600μ1 PBl加入上述离心管中，将离心管盖上盖子后，迅速地上下摇动1分 钟；
[0057] (2)再加入600μ1 ΡΒ2(含酒精），将离心管盖上盖子后，迅速地上下摇动10秒；
[0058] (3)取600μ1的混合溶液，加到纯化管柱中；
[0059] ⑷离心1分钟后，倒掉收集管内液体；
[0060] (5)加入600μ1 PB3(含酒精)到纯化管柱中；
[0061] (6)离心1分钟，倒掉收集管内液体；
[0062] ⑵加入600μ1 PB4(含酒精)到纯化管柱中；
[0063] ⑶离心1分钟，倒掉收集管内液体；
[0064] (9)再离心3分钟，除去残留乙醇；
[0065] (10)丢掉收集管，将纯化管柱套至新的离心管上；
[0066] (11)在纯化管柱中加入50μ1的PB5,放置在室温下1分钟；
[0067] (12)离心1分钟后，丢弃纯化管柱，所得核酸萃取物可以立即使用，或存放在4°C下 1小时内用完。
[0068] 三、PCR 检测
[0069] 采用绝热恒温聚合酶链反应（iiPCR)技术，在特殊设计的毛细管针对单点进行恒 温加热，然后利用荧光检测系统对样品核酸进行检测分析，并在45分钟内获得检测结果。
[0070] 实验仪器：
[0071] POCKIT Micro荧光PCR核酸分析仪(瑞基海洋生物科技股份有限公司）。目的基因：
[0072] 禽流感病毒H7亚型HA基因。
[0073] 标准品：
[0074] 包含禽流感病毒H7亚型HA基因或该基因片段的重组质粒。
[0075] iiPCR反应体系如下表3所示。
[0076] 表3 iiPCR反应体系
[0077]
[0078] 引物与探针：
[0079] 引物l序列：5，-GCTTCGGGGCATCATGTT-3，（SEQIDN0:l);
[0080] 引物2序列：5，-GCACCGCATGTTTCCATTCTT-3，（SEQIDN0:2);
[0081] 探针l序列：5，-ATTGCAATGGGCCTTGTC-3，（SEQIDN0:3);
[0082] 探针2序列：5，-ATTGCAATGGGATTGGTT-3，（SEQIDN0:4)。
[0083] 检测步骤：
[0084] 1、标准品制备
[0085] 初次使用干燥标准品先用IOOyl标准品回溶液回溶，回溶后的标准品溶液在4°C条 件下保存。
[0086] 2、样本检测
[0087] (1)按照上表3所示的体系组成制备iiPCR反应液；
[0088] (2)向三个装有上述iiPCR反应液的PCR反应管中分别加入5μ1检测样本核酸萃取 物、阴性对照样本核酸萃取物和标准品溶液，利用移液器反复吹吸混合均匀；
[0089] (3)分别吸取50μ1上述混合物移入R-tubeTM;
[0090] ⑷将R-tubeTM盖上盖子后，利用微型离心机将R-tubeTM里的液体离心至管底；
[0091] (5)启动检测仪，待检测仪完成自检后将R-tubeTM放入反应槽中；
[0092] (6)盖上机器盖子后，开机启动扩增程序并完成核酸检测，结果将于反应完成后显 示在IXD面板上。
[0093] 四、检测结果
[0094] 结果显示:检测样本核酸萃取物和标准品溶液检测结果均为“+”（阳性），阴性对照 样本核酸萃取物检测结果为“一”（阴性），说明利用本检测系统可对H7亚型禽流感病毒进行 快速检测，检测结果准确，特异性好。
[0095] 实施例5禽流感病毒H7亚型检测系统灵敏度测试
[0096] —、检测对象
[0097] 将实施例4中包含禽流感病毒H7亚型HA基因片段的重组质粒进行测序，测序结果 正确的质粒采用似11〇01'(^2000精确定量，然后根据质粒分子量计算摩尔量，制备出100、33、 10和〇拷贝AU的标准品用于灵敏度测试。
[0098] 二、检测方法
[0099] iiPCR反应体系与检测方法同实施例4。
[0100] 三、测试结果
[0101] 测试结果如下表4所示。
[0102] 表4禽流感病毒H7亚型检测系统灵敏度测试
[0103]
[0104] 结果显示:本检测系统检测灵敏度可达到10拷贝（4/4)。
[0105] 以上对本发明优选的具体实施方式和实施例作了详细说明，但是本发明并不限于 上述实施方式和实施例，在本领域技术人员所具备的知识范围内，还可以在不脱离本发明 构思的前提下作出各种变化。
[0106] 序列表 <110>金瑞鸿捷（厦门）生物科技有限公司、北京华安瑞基生物科技有限公司 <120>基于POCKIT Micro荧光PCR平台的禽流感病毒H7亚型检测试剂及应 用 <130> 2017 <160 4 <170> PatentIn version 3 .3 <210> 1 <211> 18 <212> DNA <213> 入下序列 <400> 1 gcttcgggge atcatgtt 18: <210> 2 <211> 21 <212> DNA <213>人工序列 <400> 2 gcaccgcalg UiccaUct I 21 <210> 3 <21ί> i8
[0107] <212> DNA <213>入工序列 <400> 3 attgcaatgg gccttgtc 18 <210> 4 <211> IB <212> DNA <213>人丁序列 < 400> 4 attgcaatgg gattggtt 18
Claims (10)
1. 一种基于POCKIT Micro荧光PCR平台的禽流感病毒H7亚型检测试剂，其特征在于:所 述检测试剂中包含检测禽流感病毒H7亚型的特异性引物和探针，其中，所述特异性引物的 核苷酸序列如SEQ ID NO: 1和SEQ ID N0:2所示，所述探针的核苷酸序列如SEQ ID N0:3和 SEQ ID NO:4所示，所述探针的核苷酸序列的5’端连接有荧光报告基团FAM标记，3’端连接 有非荧光淬灭基团和MGB修饰基团。
2. 如权利要求1所述的检测试剂在制备禽流感病毒H7亚型检测试剂盒中的应用。
3. 如权利要求1所述的检测试剂在制备禽流感病毒H7N7或H7N9亚型检测试剂盒中的应 用。
4. 如权利要求1所述的检测试剂在制备H7亚型禽流感诊断或治疗药物中的应用。
5. —种基于POCKIT Micro荧光PCR平台的禽流感病毒H7亚型检测试剂盒，其特征在于： 所述检测试剂盒中包含标准品和荧光定量PCR反应试剂;所述荧光定量PCR反应试剂包含 dNTPs、Taq酶、缓冲液、特异性引物和探针；其中，所述特异性引物的核苷酸序列如SEQ ID N0:1和SEQ ID N0:2所示，所述探针的核苷酸序列如SEQ ID N0:3和SEQ ID N0:4所示，所述 探针的核苷酸序列的5 ’端连接有荧光报告基团FAM标记，3 ’端连接有非荧光淬灭基团和MGB 修饰基团。
6. 如权利要求5所述的检测试剂盒，所述检测试剂盒中还包含阳性对照品和/或阴性对 照品。
7. 如权利要求5所述的检测试剂盒，其中所述标准品为包含禽流感病毒H7亚型HA基因 或该基因片段的重组质粒。
8. 如权利要求5所述的检测试剂盒，其中所述荧光定量PCR反应试剂为冷冻干燥粉，所 述检测试剂盒中还包含CldH2O。
9. 一种基于POCKIT Micro荧光PCR平台的禽流感病毒H7亚型检测系统，其特征在于:所 述检测系统中包含核酸萃取试剂盒、检测试剂盒、POCKIT Micro荧光PCR核酸分析仪、微型 离心机和移动电源； 所述检测试剂盒中包含标准品和荧光定量PCR反应试剂;所述荧光定量PCR反应试剂包 含dNTPs、Taq酶、缓冲液、特异性引物和探针;其中，所述特异性引物的核苷酸序列如SEQ ID N0:1和SEQ ID N0:2所示，所述探针的核苷酸序列如SEQ ID N0:3和SEQ ID N0:4所示，所述 探针的核苷酸序列的5 ’端连接有荧光报告基团FAM标记，3 ’端连接有非荧光淬灭基团和MGB 修饰基团。
10. 如权利要求9所述的检测系统，其中所述检测试剂盒中包含的标准品为包含禽流感 病毒H7亚型HA基因或该基因片段的重组质粒;所述荧光定量PCR反应试剂为冷冻干燥粉，所 述检测试剂盒中还包含CldH2O。
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