集成式微流控液滴数字化等温扩增用于尿路感染细菌快速检测

引用本文: 邹晶晶, 黎柱均, 刘云帆, 欧阳秀酝, 王羽, 蔡东洋, 何小维, 刘大渔. 集成式微流控液滴数字化等温扩增用于尿路感染细菌快速检测[J]. 分析化学, 2022, 50(8): 1158-1167,1187. doi: 10.19756/j.issn.0253-3820.221085 shu

Citation: ZOU Jing-Jing, LI Zhu-Jun, LIU Yun-Fan, OUYANG Xiu-Yun, WANG Yu, CAI Dong-Yang, HE Xiao-Wei, LIU Da-Yu. Integrated Microfluidic Droplet Digital Isothermal Amplification Enabled Rapid Detection of Urinary Tract Infection Bacteria[J]. Chinese Journal of Analytical Chemistry, 2022, 50(8): 1158-1167,1187. doi: 10.19756/j.issn.0253-3820.221085 shu

集成式微流控液滴数字化等温扩增用于尿路感染细菌快速检测

邹晶晶 ^1, ,
黎柱均 ^2, ,
刘云帆 ^3, ,
欧阳秀酝 ^1, ,
王羽 ^2, ,
蔡东洋 ^2, ,
何小维 ^{1,
,} ,
刘大渔 ^2,

1.
华南理工大学食品科学与工程学院, 广州 510640;
2.
华南理工大学附属第二医院检验科, 广州 510180;
3.
桂林电子科技大学数学与计算科学学院, 桂林 541004

通讯作者: 何小维,E-mail:fexwhe@scut.edu.cn

基金项目:
中国博士后科学基金项目(Nos.2020M682663, 2021M690780)、广东省自然科学基金项目(Nos.2020A1515010935, 2020A1515010754, 2020A1515110853, 2021A1515010182)、广州市第一人民医院红棉计划项目(No.PT81871726)和广东省即时检测(POCT)企业重点实验室项目(No.2021B1212050016)资助。

摘要: 数字化核酸分析技术具有不依赖标准品的绝对定量分析能力,因而有利于实现病原分子的快速诊断。现有的数字化核酸分析方法通常采用离线式分析,操作繁琐且耗时较长,难以满足医疗资源有限情况下的检测需求。本研究建立了一种集成式微流控液滴数字化等温扩增方法。采用的微流控芯片集成了序列液滴核酸提取、负压驱动液滴生成和液滴环介导等温扩增(LAMP)功能,可采用集成化方式在1.5 h内完成细菌核酸数字化分析。此微流控芯片对大肠埃希氏菌的核酸提取效率为93.68%±32.38%;使用注射器负压驱动可在4 min内生成约20000个液滴,液滴体积的相对标准偏差(RSD)小于10%;液滴数字化LAMP检测动态范围跨越4个数量级(2.36×10⁴~1.71×10⁷ CFU/mL)。利用本方法检测尿路感染临床标本(n=13)中的大肠埃希菌,与传统定量培养方法结果相比较,本方法的检测灵敏度和特异性均为100%(K_appa=1,p＜0.01)。本方法具有操作简便、定量分析准确的优点,为病原体的现场快速检测提供了一种有力的工具。

关键词:

English

Integrated Microfluidic Droplet Digital Isothermal Amplification Enabled Rapid Detection of Urinary Tract Infection Bacteria

1.
College of Food Science and Engineering, South China University of Technology, Guangzhou 510640, China;
2.
Department of Clinical Laboratory, The Second Affiliated Hospital of South China University of Technology, Guangzhou 510180, China;
3.
School of Mathematics and Computing Science, Guilin University of Electronic Technology, Guilin 541004, China

Corresponding author: HE Xiao-Wei, fexwhe@scut.edu.cn

Received Date: 21 February 2022
Revised Date: 10 May 2022
Fund Project:
Supported by the China Postdoctoral Science Foundation (Nos.2020M682663, 2021M690780), the Natural Science Foundation of Guangdong Province,China (Nos.2020A1515010935, 2020A1515010754, 2020A1515110853, 2021A1515010182), the Science Foundation of Guangzhou First People's Hospital (No.PT81871726) and the Guangdong Provincial Key Laboratory of Point-of-care Testing (POCT) (No.2021B1212050016).

Abstract: Digital nucleic acid analysis is a favorable tool for rapid molecular detection of pathogens due to its absolute quantification capability without the necessity of standard curves. However, current digital nucleic acid analysis platforms commonly perform the workflow with discrete facilities, which complicates the operation process and lengthens the turnaround time, therefore restricting its adoption in resource-limited settings. To address this issue, an integrated microfluidic droplet digital isothermal amplification system was developed in this work. This system integrated sequential droplets-based nucleic acid extraction unit, syringe-vacuum actuated droplet generation unit, and droplet digital loop-mediated isothermal amplification (LAMP) unit, achieving digital analysis of bacterial nucleic acid within 1.5 h in an integrated manner. The nucleic acid extraction unit could complete E.coli genomic DNA extraction with the efficiency of 93.68%±32.38%, the droplet generation unit could produce 20000 droplets in 4 min with the relative standard deviation (RSD) less than 10%. The LAMP reaction could be performed with a linear dynamic range of 4 orders of magnitude (2.36×10⁴-1.71×1 0⁷ CFU/mL). Compared to traditional culture method, the results obtained from analysis of E.coli in UTI clinical samples (n=13) showed that both the detection sensitivity and specificity of this digital nucleic acid analysis method were 100% (K_appa=1, p<0.01). With the advantages of accurate quantification and ease of operation, this integrated microfluidic droplet digital isothermal amplification system was expected to be a favorable tool for rapid point-of-care detection of pathogens in resource-limited settings.

Key words:

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1.
沈阳化工大学材料科学与工程学院沈阳 110142

集成式微流控液滴数字化等温扩增用于尿路感染细菌快速检测

通讯作者: 何小维,E-mail:fexwhe@scut.edu.cn

English

Integrated Microfluidic Droplet Digital Isothermal Amplification Enabled Rapid Detection of Urinary Tract Infection Bacteria

Corresponding author: HE Xiao-Wei, fexwhe@scut.edu.cn

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通讯作者: 陈斌, bchen63@163.com

中国化学会秘书处

集成式微流控液滴数字化等温扩增用于尿路感染细菌快速检测

通讯作者: 何小维,E-mail:fexwhe@scut.edu.cn

English

Integrated Microfluidic Droplet Digital Isothermal Amplification Enabled Rapid Detection of Urinary Tract Infection Bacteria

Corresponding author: HE Xiao-Wei, fexwhe@scut.edu.cn

CCS Chemistry：嵌段共聚物自组装成 “三明治”二维有机材料，实现纳米级压力传感功能

CCS Chemistry：颜徐州、鲍哲南： 你的皮肤可能是一片SPMs

CCS Chemistry：工作高效不疲劳，只须让催化剂动起来

CCS Chemistry：静电组装导向聚合- 聚电解质纳米凝胶量化制备新策略

CCS Chemistry：金黄色葡萄球菌醛基脱氢酶是如何识别特异性底物的？

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