CN116287333A - 一种检测脓毒症病原体的微流控芯片及试剂盒 - Google Patents

一种检测脓毒症病原体的微流控芯片及试剂盒 Download PDF

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CN116287333A
CN116287333A CN202310050690.0A CN202310050690A CN116287333A CN 116287333 A CN116287333 A CN 116287333A CN 202310050690 A CN202310050690 A CN 202310050690A CN 116287333 A CN116287333 A CN 116287333A
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sepsis
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陈奇通
李文放
袁晓伟
朱妍
郦奕帆
张丹颖
邹知静
杨帆
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Shanghai Changzheng Hospital
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Abstract

本发明涉及生物检测领域，提供一种检测脓毒症病原体的微流控芯片及试剂盒；包括：进样孔以及若干反应检测孔；若干反应检测孔内预埋有脓毒症病原体的引物和探针；其中，脓毒症病原体包括：大肠埃希菌、铜绿假单胞菌、肺炎克雷伯菌、鲍曼不动杆菌、流感嗜血杆菌、嗜麦芽窄食单胞菌、阴沟杆菌、金黄色葡萄球菌、肺炎链球菌、溶血链球菌、肠球菌、淋病奈瑟菌、脑膜炎球菌、念珠菌、曲霉菌、假丝酵母菌、隐球菌；本发明将多种革兰氏阴性细菌、革兰氏阳性细菌以及真菌所对应的探针集合在同一块微流控芯片上，可以同时全面检测多种脓毒症常见病原体，节省了培养时间以及成本，具有较高的临床应用前景。

Description

一种检测脓毒症病原体的微流控芯片及试剂盒
技术领域
本发明涉及生物检测领域，尤其涉及一种检测脓毒症病原体的微流控芯片及试剂盒。
背景技术
脓毒症是感染引起宿主反应失调，导致危及生命的器官功能损害的症候群，是一个高病死率的临床综合征。脓毒症不仅严重威胁人类健康，也给医疗卫生带来了巨大的经济负担。
微流控指的是使用微管道处理或操纵微小流体的系统所涉及的科学和技术，是一门涉及化学、流体物理、微电子、新材料、生物学和生物医学工程的新兴交叉学科，因其具有微型化、集成化等特征，微流控装置通常被称为微流控芯片，也被称为芯片实验室和微全分析系统。微流控的早期概念可以追溯到19世纪70年代采用光刻技术在硅片上制作的气相色谱仪，而后又发展为微流控毛细管电泳仪和微反应器等，其重要特征之一是微尺度环境下具有独特的流体性质，如层流和液滴等，借助这些独特的流体现象，微流控可以实现一系列常规方法所难以完成的微加工和微操作。
近年来，微流控芯片结合便携式、全自动的分析检测仪器形成微流控即时检测系统，广泛应用于食品安全检测、环境监测、医疗生化诊断等领域，实现现场、精准、快速检测。对比传统的化学、生物分析检测方法，微流控芯片分析平台具有“多、快、准、省”(一次性可以检测多个样本或多个指标、15分钟内快速获取检测结果、全自动操作、试剂消耗量降为常规检测的百分之一甚至千分之一、生产及检测成本低)的显著优势。
发明内容
本发明的目的是针对现有技术中的不足，提供一种检测脓毒症病原体的微流控芯片及试剂盒。
为实现上述目的，本发明采取的技术方案是：
本发明的第一方面是提供一种检测脓毒症病原体的微流控芯片，包括：进样孔以及若干反应检测孔；若干所述反应检测孔内预埋有所述脓毒症病原体的引物和探针；
其中，所述脓毒症病原体包括：大肠埃希菌、铜绿假单胞菌、肺炎克雷伯菌、鲍曼不动杆菌、流感嗜血杆菌、嗜麦芽窄食单胞菌、阴沟杆菌、金黄色葡萄球菌、肺炎链球菌、溶血链球菌、肠球菌、淋病奈瑟菌、脑膜炎球菌、念珠菌、曲霉菌、假丝酵母菌以及隐球菌。
优选地，所述引物和探针的核苷酸序列如SEQ ID NO：1-51所示。
更优选地，所述引物和探针分为3组，分别为：SEQ ID NO：1-21，SEQ IDNO：22-39，SEQ ID NO：40-51。
优选地，所述探针为荧光探针。
优选地，所述反应检测孔还包括：PCR反应试剂。
本发明的第二方面是提供一种含有上述微流控芯片的试剂盒，包括：样品前处理组件。
优选地，所述样品前处理组件采用磁珠法提取核酸。
优选地，检测样本为咽拭子、唾液、痰液、血液、尿液、脑脊液或局部感染灶分泌液。
本发明采用以上技术方案，与现有技术相比，具有如下技术效果：
本发明将多种革兰氏阴性细菌、革兰氏阳性细菌以及真菌所对应的探针集合在同一块微流控芯片上，可以同时全面检测多种脓毒症常见病原体，节省了培养时间以及成本，具有较高的临床应用前景。
具体实施方式
下面将对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有作出创造性劳动的前提下所获得的所有其他实施例，都属于本发明保护的范围。
需要说明的是，在不冲突的情况下，本发明中的实施例及实施例中的特征可以相互组合。
下面结合具体实施例对本发明作进一步说明，但不作为本发明的限定。
实施例
1、采样(以血液培养为例)
签署知情同意书后，按医疗常规操作采集人静脉血5mL置于cfDNA试管(cell-freeDNA tube)中，在室温下进行第一次预处理，3000转(约2400g)，离心10分钟，取上清1mL置于BP管中，接着进行第二次离心，将BP管在室温下10000转(约9000g)，离心10分钟，取上清0.9mL至冻存管中，制备成等离子体，使用QIAamp循环核酸试剂盒(QIAGEN，Valencia，CA，USA)从血浆中提取游离DNA(cfDNA)。
2、PCR检测
反应所用引物及探针序列如下：
Figure BDA0004057785470000031
Figure BDA0004057785470000041
Figure BDA0004057785470000051
Figure BDA0004057785470000061
PCR反应体系：
Figure BDA0004057785470000062
Figure BDA0004057785470000071
综上所述，本发明将多种革兰氏阴性细菌、革兰氏阳性细菌以及真菌所对应的探针集合在同一块微流控芯片上，可以同时全面检测多种脓毒症常见病原体，节省了培养时间以及成本，具有较高的临床应用前景。
以上所述仅为本发明较佳的实施例，并非因此限制本发明的实施方式及保护范围，对于本领域技术人员而言，应当能够意识到凡运用本发明说明书内容所作出的等同替换和显而易见的变化所得到的方案，均应当包含在本发明的保护范围内。

Claims (8)

1.一种检测脓毒症病原体的微流控芯片，其特征在于，包括：进样孔以及若干反应检测孔；若干所述反应检测孔内预埋有所述脓毒症病原体的引物和探针；
其中，所述脓毒症病原体包括：大肠埃希菌、铜绿假单胞菌、肺炎克雷伯菌、鲍曼不动杆菌、流感嗜血杆菌、嗜麦芽窄食单胞菌、阴沟杆菌、金黄色葡萄球菌、肺炎链球菌、溶血链球菌、肠球菌、淋病奈瑟菌、脑膜炎球菌、念珠菌、曲霉菌、假丝酵母菌以及隐球菌。
2.根据权利要求1所述的微流控芯片，其特征在于，所述引物和探针的核苷酸序列如SEQ ID NO：1-51所示。
3.根据权利要求2所述的微流控芯片，其特征在于，所述引物和探针分为3组，分别为：SEQ ID NO：1-21，SEQ ID NO：22-39，SEQ ID NO：40-51。
4.根据权利要求1所述的微流控芯片，其特征在于，所述探针为荧光探针。
5.根据权利要求1所述的微流控芯片，其特征在于，所述反应检测孔还包括：PCR反应试剂。
6.一种含有权利要求1-5任一项所述微流控芯片的试剂盒，其特征在于，包括：样品前处理组件。
7.根据权利要求6所述的试剂盒，其特征在于，所述样品前处理组件采用磁珠法提取核酸。
8.根据权利要求6所述的试剂盒，其特征在于，检测样本为咽拭子、唾液、痰液、血液、尿液、脑脊液或局部感染灶分泌液。
CN202310050690.0A 2023-02-01 2023-02-01 一种检测脓毒症病原体的微流控芯片及试剂盒 Pending CN116287333A (zh)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117491284A (zh) * 2023-11-03 2024-02-02 上海长征医院 一种基于微流控技术的脓毒症即时检测设备

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117491284A (zh) * 2023-11-03 2024-02-02 上海长征医院 一种基于微流控技术的脓毒症即时检测设备
CN117491284B (zh) * 2023-11-03 2024-05-07 上海长征医院 一种基于微流控技术的脓毒症即时检测设备

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