通过基于微流控芯片的环介导等温扩增快速检测引起下呼吸道感染的细菌病原体
1Laboratory Medicine, Guangdong Provincial People’s Hospital (Guangdong Academy of Medical Sciences),Southern Medical University, 2Department of Laboratory Medicine,Shengli Oilfield Central Hospital, 3Guangdong Cardiovascular Institute, Guangdong Provincial People’s Hospital (Guangdong Academy of Medical Sciences),Southern Medical University, 4Dermatology Hospital,Southern Medical University
Summary
各种细菌病原体如果不准确发现和及时治疗，会导致呼吸道感染并导致严重的健康问题。 通过环介导的等温扩增快速准确地检测这些病原体，可在临床环境中有效管理和控制呼吸道感染。
Abstract
呼吸道感染 （RTI） 是临床环境中最常见的问题之一。快速准确地鉴定细菌病原体将为管理和治疗RTIs提供实用指南。本研究描述了一种通过多通道环介导的等温扩增 （LAMP） 快速检测引起呼吸道感染的细菌病原体的方法。LAMP是一种灵敏、特异的诊断工具，可快速检测细菌核酸，准确度高，可靠性高。与传统的细菌培养方法相比，所提出的方法具有显着的优势，传统的细菌培养方法非常耗时，并且通常需要更高的灵敏度来检测低水平的细菌核酸。本文介绍了利用LAMP检测下呼吸道样本（痰液、支气管灌洗液和肺泡灌洗液）的肺炎 克雷伯菌 感染及其多种合并感染的代表性结果。综上所述，多通道LAMP方法提供了一种快速有效的方法，用于识别临床样本中的单一和多种细菌病原体，有助于防止细菌性病原体的传播，并有助于RTIs的适当治疗。
Introduction
由细菌病原体引起的呼吸道感染（RTIs）是全球发病率和死亡率的主要原因1.它被定义为任何上呼吸道或下呼吸道症状，伴有持续 2-3 天的发烧。虽然上呼吸道感染比下呼吸道感染更常见，但慢性和复发性呼吸道感染也是常见的临床疾病，对个人构成巨大风险，并给医疗保健系统带来沉重负担2.RTIs的常见细菌性病原体包括肺炎链球菌3型、流感嗜血杆菌4型、金黄色葡萄球菌、大肠杆菌、肺炎克雷伯菌、麦芽窄食单胞菌等。这些致病菌通常定植在宿主鼻咽和上呼吸道的粘膜表面，引起喉咙痛和支气管炎等 RTI 的典型症状。当它们从上呼吸道扩散到下呼吸道的无菌区域时，它们会引起肺炎，并可能通过呼吸道在人与人之间传播5.在严重的情况下，它们还可能导致侵袭性细菌性疾病，尤其是菌血症性肺炎、脑膜炎和败血症，这些都是全球所有年龄组人群发病和死亡的主要原因。
传统的 RTI 检测包括使用咽拭子和痰液呼吸道样本进行微生物培养6.此外，酶联免疫吸附测定 （ELISA） 等血清学检测可检测血清中的抗体或抗原，而凝集试验可观察抗体和抗原的凝集反应以检测感染7。微生物培养被认为是诊断 RTI 的金标准，但其培养阳性率低、可靠性差和检测周期长限制了诊断效率8。实际上，快速准确地诊断RTIs对于精确根除细菌病原体至关重要。快速有效的检测方法有助于降低病原体的传播率，缩短感染持续时间，并减少不必要的抗生素使用9,10。基于分子生物学的方法大大加快了检测速度，例如聚合酶链反应 （PCR），它通过扩增靶基因的 DNA 序列来检测病原体。然而，传统的PCR需要复杂的温度循环设备，既繁琐又耗时。此外，使用PCR进行的每次DNA扩增（实时荧光定量PCR除外）都以电泳分离产物结束，这也需要时间。产品的可视化需要染料，其中许多染料具有致突变性或致癌性。因此，不断开发诊断RTI细菌病原体的新方法和技术势在必行。
环介导的等温扩增（LAMP）是一种新型的分子技术，最初由Notomi等人于2000年开发11。LAMP可以在稳定的等温条件下扩增DNA，无需复杂的温度循环设备，使其适用于快速检测，降低了设备的复杂性和成本12。LAMP 可以高灵敏度检测低浓度的靶标 DNA13。它使用多种特异性引物来提高靶序列的选择性并减少假阳性的可能性14。LAMP因其易用性、速度和直观的操作而逐渐在临床实验室中得到广泛使用，甚至用于检测RTI。在这项研究中，我们研究了 LAMP 在检测临床样本（痰液、支气管灌洗液和肺泡灌洗液）中较低 RTI 的有效性，如图 1 所示。很明显，与传统测试相比，LAMP在较低的RTI检测方面具有速度、灵敏度和易用性等优势，使其成为一个有前途的应用。
图 1：LAMP 检测方法示意图。 请点击这里查看此图的较大版本.
Protocol
本研究所有样本均经广东省人民医院伦理审查委员会（批准文号：KY2023-1114-01）审定通过。所有参与者在实验前签署了书面知情同意书。用于研究的试剂和设备列在 材料表中。协议中使用的缩写列在 补充表1中。 1.采集下呼吸道临床样本 痰液收集用清水清洁口腔和牙齿，如果适用，请确保摘下假牙。用力咳嗽，将深呼吸道痰…
Representative Results
该实验采用等温放大技术，在微流控圆盘芯片上进行反应。该反应在微流控芯片核酸分析仪上进行，采用荧光染料插入方法。等温反应在65°C的恒温下进行，同时进行实时荧光分析。阳性样品在具有链置换官能团的聚合酶作用下进行扩增，产生S形扩增曲线。这个一步法完成了靶基因的扩增和检测。本研究选取了7个实验结果不同的样品，验证了实验设计的可靠性。大写字母 S 代表样本。S1 是感染单…
Discussion
呼吸道感染是普遍存在的医院相关感染，会给患者带来严重后果，并使死亡率不断上升16。及时准确地识别潜在的病原体，然后使用有效的抗生素是成功治疗和改善预后的关键，特别是考虑到传统培养方法固有的局限性17.在这项研究中，我们使用基于 LAMP 的方法来确定单个或多个感染，以快速准确地检测 RTI。该快速检测系统是通过将LAMP技术与微流控技术相结合…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们非常感谢广东省基础与应用基础研究基金（批准号：2022A1515220023）和广东省人民医院高级人才研究基金（批准号：2022A1515220023）提供资金支持。KY012023293）。
Materials
| Bath Incubator(MK2000-2) | ALLSHENG | Provide a constant temperature environment | |
| Bronchial lavage fluid collector head | TIANPINGHUACHANG | SEDA 20172081375 | Collecting bronchoalveolar lavage fluid |
| Fiberoptic bronchoscope | OLYMPUS | SEDA 20153062703 | A flexible bronchoscope equipped with a fiberoptic light source and camera, to visually examine the airways and structures within the lungs. Assist in collecting bronchoalveolar lavage |
HR1500- B2 |
Haier | SEDA 20183541642 | Biosafety cabinet |
| NAOH | MACKLIN | S817977 | Liquefy viscous lower respiratory tract sample |
| Nucleic acid detection kit for respiratory tract pathogens | Capitalbio Technology | SEDA 20173401346 | Testing for bacteria infection |
| Nucleic acid extraction reagent | Capitalbio Technology | SEDA 20160034 | For DNA extraction |
| RTisochip-W | Capitalbio Technology | SEDA 20193220539 | Loop-mediated Isothermal Amplification |
| THERMO ST16R | Thermo Fisher Scientific | SEDA 20180585 | Centrifuge the residual liquid off the wall of the tube. |
| Vortex mixer VM-5005 | JOANLAB | For mixing reagent |
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Cite This Article
Lai, J., Qin, Y., Liao, Y., Si, Y., Yuan, Q., Huang, S., Tang, Y., Wang, J., Wang, L. Rapid Detection of Bacterial Pathogens Causing Lower Respiratory Tract Infections via Microfluidic-Chip-Based Loop-Mediated Isothermal Amplification. J. Vis. Exp. (205), e66677, doi:10.3791/66677 (2024).