Name: Author Spotlight: Advancing Rapid Detection of Respiratory Pathogens Using Microfluidic Chip
Uploaded: 2024-03-29T14:50:00
Description: Read this Scientific Article Protocol about Advancing Rapid Detection of Respiratory Pathogens Using Microfluidic Chip at JoVE.com

We greatly appreciated the financial support provided by the Guangdong Basic and Applied Basic Research Foundation (Grant No. 2022A1515220023) and the Research Foundation for Advanced Talents of Guandong Provincial People&#39;s Hospital (Grant No. KY012023293).

All samples for this study were evaluated and approved by the Ethics Review Committee of Guangdong Provincial People&#39;s Hospital (Approval Number: KY2023-1114-01). All participants signed written informed consent before the experiments. The reagents and equipment used for the study are listed in the Table of Materials. The abbreviations used in the protocol are listed in Supplementary Table 1.
1. Collection of clinical samples from the lower respirato...

Human Respiratory Bacterial Infection Diagnosis Using Isothermal Amplification

<ol>
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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+basics+and+the+advancements+in+diagnosis+of+bacterial+lower+respiratory+tract+infections.">The basics and the advancements in diagnosis of bacterial lower respiratory tract infections.</a> Diagnostics (Basel). 9 (2), 37 (2019).</li><li>Hanson, K. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+testing+for+acute+respiratory+tract+infections:+Clinical+and+diagnostic+recommendations+from+the+IDSA's+diagnostics+committee.">Molecular testing for acute respiratory tract infections: Clinical and diagnostic recommendations from the IDSA's diagnostics committee.</a> Clin Infect Dis. 71 (10), 2744-2751 (2020).</li><li>Daniel Reynolds, J. P. 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O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loop-mediated+isothermal+amplification+of+DNA.">Loop-mediated isothermal amplification of DNA.</a> Nucleic Acids Res. 28 (12), 63 (2000).</li><li>Soroka, M., Wasowicz, B., Rymaszewska, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loop-mediated+isothermal+amplification+(LAMP):+The+better+sibling+of+PCR.">Loop-mediated isothermal amplification (LAMP): The better sibling of PCR.</a> Cells. 10 (8), 1931 (2021).</li><li>Mori, Y., Kitao, M., Tomita, N., Notomi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+turbidimetry+of+lamp+reaction+for+quantifying+template+DNA.">Real-time turbidimetry of lamp reaction for quantifying template DNA.</a> Biophys J. 59 (2), 145-157 (2004).</li><li>Parida, M., Sannarangaiah, S., Dash, P. 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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loop-mediated+isothermal+amplification+technology:+Towards+point+of+care+diagnostics.">Loop-mediated isothermal amplification technology: Towards point of care diagnostics.</a> PLoS Negl Trop Dis. 6 (6), e1572 (2012).</li><li>Notomi, T., Mori, Y., Tomita, N., Kanda, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loop-mediated+isothermal+amplification+(LAMP):+Principle,+features,+and+future+prospects.">Loop-mediated isothermal amplification (LAMP): Principle, features, and future prospects.</a> J Microbiol. 53 (1), 1-5 (2015).</li><li>Ajibola, O., Gulumbe, B., Eze, A., Obishakin, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tools+for+detection+of+schistosomiasis+in+resource+limited+settings.">Tools for detection of schistosomiasis in resource limited settings.</a> Medical Sciences. 6 (2), 39 (2018).</li><li>Kumar, Y. 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Respiratory tract infections are prevalent hospital-associated infections, imposing severe consequences on patients and escalating mortality rates16. Timely and accurately identifying potential pathogens followed by effective antibiotics is the key to successful treatment and improving prognosis, particularly given the limitations inherent in traditional culture methods17. In this study, we used a LAMP-based method to determine single or multiple infections for fast and pre...

Respiratory tract infections (RTIs) caused by bacterial pathogens primarily contribute to morbidity and mortality worldwide1. It is defined as any upper or lower respiratory symptoms accompanied by fever lasting 2-3 days. While upper respiratory infection is more common than lower respiratory infection, chronic and recurrent respiratory tract infections are also common clinical conditions, posing great risks to individuals and placing a significant burden on healthcare systems2. Common bacterial pathogens of RTIs include Streptococcus pneumoniae3, Haemophilus influenzae4, Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Stenotrophomonas maltophilia, among others. These pathogenic bacteria usually colonize the mucosal surfaces of the host&#39;s nasopharynx and upper respiratory tract, causing typical symptoms of RTIs such as sore throat and bronchitis. They cause pneumonia when they spread from the upper respiratory tract to sterile areas of the lower respiratory tract and may spread from person to person through the respiratory tract5. In severe cases, they can also lead to invasive bacterial diseases, especially bacteremic pneumonia, meningitis, and sepsis, which are leading causes of morbidity and mortality in people of all age groups worldwide.
Traditional tests for RTIs involve microbiological culture using throat swabs and sputum respiratory samples6. Additionally, serological tests such as enzyme-linked immunosorbent assay (ELISA) detect antibodies or antigens in serum, while agglutination tests observe the agglutination reaction of antibodies and antigens to detect infection7. Microbial culture is considered the gold standard for diagnosing RTIs, but its low culture positivity rate, poor reliability, and long detection cycle limit diagnostic efficiency8. In reality, rapid and accurate diagnosis of RTIs is crucial for precise eradication of the bacterial pathogen. Quick and effective detection methods can help reduce the transmission rate of pathogens, shorten the duration of infection, and decrease unnecessary antibiotic use9,10. Molecular biology-based methods significantly expedite detection, such as polymerase chain reaction (PCR), which amplifies a target gene&#39;s DNA sequence to detect pathogens. However, traditional PCR necessitates complex temperature cycling equipment, which is cumbersome and time-consuming. Furthermore, every DNA amplification using PCR (except real-time PCR) concludes with electrophoretic separation of the product, which also takes time. Visualization of the product requires dyes, many of which are mutagenic or carcinogenic. Therefore, it is imperative to continuously develop new methods and technologies for diagnosing RTI bacterial pathogens.
Loop-Mediated Isothermal Amplification (LAMP) is a novel and emerging molecular technology initially developed by Notomi et al. in 200011. LAMP can amplify DNA under stable isothermal conditions without complex temperature cycling equipment, which makes it suitable for rapid detection and reduces equipment complexity and cost12. LAMP can detect low concentrations of target DNA with high sensitivity13. It uses multiple specific primers to improve selectivity for target sequences and reduce the possibility of false positives14. LAMP is gradually being widely used in clinical laboratories due to its ease, speed, and intuitive operation, even for detecting RTIs. In this study, we investigated the effectiveness of LAMP in detecting lower RTIs in clinical samples (sputum, bronchial lavage fluid, and alveolar lavage fluid), as shown in Figure 1. It is evident that LAMP offers advantages such as speed, sensitivity, and ease of use over traditional tests in lower RTI detection, making it a promising application.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66677/66677fig01.jpg" /> 
Figure 1: Schematic illustration of the LAMP detection method. <a href="https://www.jove.com/files/ftp_upload/66677/66677fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Bath Incubator(MK2000-2)</td>
			<td>ALLSHENG</td>
			<td></td>
			<td>Provide a constant temperature environment</td>
		</tr>
		<tr>
			<td>Bronchial lavage fluid collector head</td>
			<td>TIANPINGHUACHANG</td>
			<td>SEDA 20172081375</td>
			<td>Collecting bronchoalveolar lavage fluid</td>
		</tr>
		<tr>
			<td>Fiberoptic bronchoscope</td>
			<td>OLYMPUS</td>
			<td>SEDA 20153062703</td>
			<td>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</td>
		</tr>
		<tr>
			<td>HR1500-<img alt="Equation 1" src="/files/ftp_upload/66677/66677eq01v2.jpg" style="margin: auto;" />B2</td>
			<td>Haier</td>
			<td>SEDA 20183541642</td>
			<td>Biosafety cabinet</td>
		</tr>
		<tr>
			<td>NAOH</td>
			<td>MACKLIN</td>
			<td>S817977</td>
			<td>Liquefy viscous lower respiratory tract sample</td>
		</tr>
		<tr>
			<td>Nucleic acid detection kit for respiratory tract pathogens</td>
			<td>Capitalbio Technology</td>
			<td>SEDA 20173401346</td>
			<td>Testing for bacteria infection</td>
		</tr>
		<tr>
			<td>Nucleic acid extraction reagent</td>
			<td>Capitalbio Technology</td>
			<td>SEDA 20160034</td>
			<td>For DNA extraction</td>
		</tr>
		<tr>
			<td>RTisochip-W</td>
			<td>Capitalbio Technology</td>
			<td>SEDA 20193220539</td>
			<td>Loop-mediated Isothermal Amplification</td>
		</tr>
		<tr>
			<td>THERMO ST16R</td>
			<td>Thermo Fisher Scientific</td>
			<td>SEDA 20180585</td>
			<td>Centrifuge the residual liquid off the wall of the tube.</td>
		</tr>
		<tr>
			<td>Vortex mixer VM-5005</td>
			<td>JOANLAB</td>
			<td></td>
			<td>For mixing reagent</td>
		</tr>
	</tbody>
</table>

rapid detection of bacterial pathogens causing lower respiratory tract infections via microfluidic-chip-based loop-mediated isothermal amplification

Respiratory tract infections (RTIs) are among the most common problems in clinical settings. Rapid and accurate identification of bacterial pathogens will provide practical guidelines for managing and treating RTIs. This study describes a method for rapidly detecting bacterial pathogens that cause respiratory tract infections via multi-channel loop-mediated isothermal amplification (LAMP). LAMP is a sensitive and specific diagnostic tool that rapidly detects bacterial nucleic acids with high accuracy and reliability. The proposed method offers a significant advantage over traditional bacterial culturing methods, which are time-consuming and often require greater sensitivity for detecting low levels of bacterial nucleic acids. This article presents representative results of K. pneumoniae infection and its multiple co-infections using LAMP to detect samples (sputum, bronchial lavage fluid, and alveolar lavage fluid) from the lower respiratory tract. In summary, the multi-channel LAMP method provides a rapid and efficient means of identifying single and multiple bacterial pathogens in clinical samples, which can help prevent the spread of bacterial pathogens and aid in the appropriate treatment of RTIs.

Author Spotlight: Advancing Rapid Detection of Respiratory Pathogens Using Microfluidic Chip 

Rapid Detection of Bacterial Pathogens Causing Lower Respiratory Tract Infections via Microfluidic-Chip-Based Loop-Mediated Isothermal Amplification

This experiment employs isothermal amplification technology, conducting reactions on a microfluidic disc chip. The reaction occurs on a microfluidic chip nucleic acid analyzer, employing a fluorescence dye insertion method. The isothermal reaction is performed at a constant temperature of 65 &#176;C, and real-time fluorescence analysis is carried out simultaneously. Positive samples undergo amplification under the action of polymerase with chain displacement functionality, resulting in an S-shaped amplification curve. Th...

Read this Scientific Article Protocol about Advancing Rapid Detection of Respiratory Pathogens Using Microfluidic Chip at JoVE.com

Various bacterial pathogens can cause respiratory tract infections and lead to serious health issues if not detected accurately and treated promptly. Rapid and accurate detection of these pathogens via&#160;loop-mediated isothermal amplification provides effective management and control of respiratory tract infections in clinical settings.

Respiratory tract infections (RTIs) are among the most common problems in clinical settings. Rapid and accurate identification of bacterial pathogens will ...