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 respiratory tract
<ol>
	<li>Sputum collection
	<ol>
		<li>Cleanse the oral cavity and teeth with clean water, ensuring denture removal if applicable. Cough forcefully to expel deep respiratory sputum into a sputum container (minimum 0.6 mL).</li>
		<li>Avoid contamination with saliva or nasal discharge. If coughing is difficult, administer inhalation of a 100 g/L NaCl solution at 45 &#176;C to facilitate expectoration. Transfer the specimen to a sterile, sealable container. 
		NOTE: Ensure the sputum samples are collected in the morning and before any antibacterial drug use.</li>
	</ol>
	</li>
	<li>Bronchial lavage fluid (BLF)
	<ol>
		<li>Insert the collector head into the trachea from the nostril or tracheostomy site (approximately 30 cm deep). Inject 5 mL of saline, establish negative pressure, rotate the collector head, and slowly withdraw.</li>
		<li>Collect the extracted mucus and rinse the collector once with a sampling solution (physiological saline or sterile water for injection). Alternatively, connect a pediatric urinary catheter to a 50 mL syringe as a substitute for collection.</li>
	</ol>
	</li>
	<li>Alveolar lavage fluid (ALF)
	<ol>
		<li>Identify the lesion location through chest imaging examinations. Select the most significant or rapidly progressing area for lavage.</li>
		<li>Inject 1-2 mL of 2% lidocaine into the bronchial segment through the biopsy channel during lavage, providing local anesthesia for the lavaged lung segment15. For patients under intravenous general anesthesia who still exhibit strong airway reactions, inject 1-2 mL of 2% lidocaine.</li>
		<li>After local anesthesia, insert a fiberoptic bronchoscope through the mouth or nose, passing through the pharynx into the bronchus of the right middle lobe or the lingual segment of the left lung. Place its tip at the opening of the bronchial branch and slowly introduce sterile physiological saline.</li>
		<li>Administer 30-50 mL saline per session, with 100-250 mL volume (max. 300 mL). Collect the extracted mucus in a sealed sterile container.</li>
	</ol>
	</li>
</ol>
2. DNA extraction
<ol>
	<li>Based on the viscosity of lower respiratory tract samples, add an appropriate amount of 10% NaOH. For samples with low viscosity, add 2-3 times the volume of the sample of liquefying solution. For samples with moderate viscosity, add 5-6 times the volume of the sample of liquefying solution. For samples with high viscosity, add 8-10 times the volume of the sample of liquefying solution.
	<ol>
		<li>Adjust the volume of 10% NaOH according to the sample viscosity. Disperse the samples as uniformly as possible using a vortex mixer for 15 s and incubate at 37 &#176;C for 30 min for liquefaction. 
		NOTE: For thicker samples, increase the volume of NaOH or extend the liquefaction time. The liquefaction quality of samples directly influences the subsequent extraction efficiency. Ideally, liquefied samples should exhibit a consistent, non-sticky consistency.</li>
	</ol>
	</li>
	<li>Pipette 1 mL of the liquefied sample into a 1.5 mL centrifuge tube. Centrifuge at a speed of 15,777 &#215; g for 5 min at 2-6 &#176;C, then use a pipette to remove and discard the supernatant. 
	NOTE: When aspirating the sample, avoid drawing impurities from the bottom of the tube; if there are many impurities, the sample can be centrifuged at 1,753-2,739 &#215; g for 1 min before aspiration.</li>
	<li>Add 1 mL of washing solution to the centrifuge tube and vortex to lift the precipitate from the bottom of the tube. There is no need to disperse it completely.
	<ol>
		<li>Centrifuge the solution at 15,777 &#215; g for 5 min, discard the supernatant, and try not to touch the precipitate. 
		NOTE: Ensure thorough removal of the washing solution to avoid affecting subsequent amplification.</li>
	</ol>
	</li>
	<li>Add 100 &#181;L of nucleic acid extraction solution to the centrifuge tube. Use a pipette to aspirate and mix the precipitate thoroughly. Transfer the liquid and precipitate together into a nucleic acid extraction tube. The composition of the nucleic acid extraction solution is presented in Table 1.</li>
	<li>Place the nucleic acid extraction tubes in a vortex mixer and vortex at medium speed for at least 5 min. After vortexing, transfer the nucleic acid extraction tubes to a metal bath with a constant temperature and heat at 100 &#176;C for 5 min.</li>
	<li>Centrifuge at 15,777 &#215; g at 2-6 &#176;C for 5 min and set aside. 
	NOTE: If the PCR amplification reaction is performed within 24 h, the nucleic acid can be stored at 4 &#176;C. After the amplification, store the nucleic acid at -20 &#176;C. For long-term storage (beyond 24 h), store the nucleic acid at -20 &#176;C. When ready to use again, thaw the sample naturally, vortex to mix, heat in a 95 &#176;C water bath for 5 min, centrifuge at 10,956 &#215; g for 1 min, and use the supernatant for PCR amplification.</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Solution</td>
			<td>Components</td>
			<td>Number</td>
			<td>Specification</td>
		</tr>
		<tr>
			<td>Washing Solution</td>
			<td>10 mM EDTA</td>
			<td>1 bottle</td>
			<td>24 mL/bottle</td>
		</tr>
		<tr>
			<td>Nucleic Acid Extraction Reagent</td>
			<td>10mM Tris-HCl, 1mM EDTA, Nucleic acid preservatives</td>
			<td>2 tubes</td>
			<td>1.2 mL/bottle</td>
		</tr>
		<tr>
			<td>Nucleic Acid Extraction Tube</td>
			<td>Glass beads</td>
			<td>1 bag</td>
			<td>24 bottles/bag</td>
		</tr>
	</tbody>
</table>
Table 1: Composition of nucleic acid extraction reagent.
3. Loop-mediated isothermal amplification and microfluid chip
<ol>
	<li>Reagent and microfluidic chip
	<ol>
		<li>Conduct isothermal amplification reactions on a microfluidic disk-shaped chip (see Table of Materials). Carry out the constant-temperature amplification at 65 &#176;C.</li>
		<li>Perform real-time fluorescence analysis using a fluorescent dye incorporation method14 on the constant-temperature amplification microfluidic chip nucleic acid analyzer. Observe an S-shaped amplification curve for positive samples using a polymerase with strand displacement functionality.</li>
	</ol>
	</li>
	<li>Loop-mediated isothermal amplification
	<ol>
		<li>Target six sequence regions with four specific primers, including two inner primers and two outer primers (provided with the LAMP kit). 
		NOTE: DNA is continuously replicated and amplified at a constant temperature using a DNA polymerase with strand displacement functionality. The reaction process involves the dumbbell-shaped template synthesis stage, cycling amplification stage, elongation, and recycling stage, ultimately forming a mixture of DNA fragments with stem-loop and cauliflower-like structures. Refer to the kit instructions for detailed information (Supplementary File 1).</li>
		<li>Add two-loop primers to the reaction system to enhance reaction efficiency, which binds to the stem-loop structures, initiating strand displacement synthesis and cyclic replication. The composition of the nucleic acid detection kit for respiratory tract pathogens is presented in Table 2. 
		NOTE: The reagent kit utilizes the LAMP method. The principle is based on the dynamic equilibrium of DNA at around 65 &#176;C, where any primer extending through base pairing at the complementary site of double-stranded DNA causes the other strand to dissociate, forming a single strand.</li>
	</ol>
	</li>
	<li>Microfluidic chip 
	NOTE: Each microfluidic chip (see Table of Materials) is equipped with 24 reaction wells numbered counterclockwise, with the inlet and outlet for well 1 corresponding to the reaction well 1 (Figure 2). A specific set of primers for amplification and detection of a particular nucleic acid target sequence is embedded and fixed in each reaction well.
	<ol>
		<li>Mix the sample DNA with the amplification reagent. Inject the mixture into the chip and distribute it to each reaction well centrifuge at 1,000 &#215; g for 30 s at room temperature. 
		NOTE: Independent isothermal amplification reactions and real-time fluorescence detection co-occur in each reaction well on the chip. If an S-shaped amplification curve is detected in a particular reaction well, the corresponding detection index for that well is positive.</li>
	</ol>
	</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Components</td>
			<td>Composition</td>
			<td>Number</td>
		</tr>
		<tr>
			<td>Chip</td>
			<td>Primers</td>
			<td>12 places</td>
		</tr>
		<tr>
			<td>Sealing Film</td>
			<td>/</td>
			<td>1 sheet</td>
		</tr>
		<tr>
			<td>Isothermal Amplification Reagent</td>
			<td>Fluorescent dye, Enzyme</td>
			<td>270 &#181;L/tube</td>
		</tr>
		<tr>
			<td>Positive Control</td>
			<td>Escherichia coli Genomic DNA</td>
			<td>160 &#181;L/tube</td>
		</tr>
	</tbody>
</table>
Table 2: Composition of nucleic acid detection kit for respiratory tract pathogens.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66677/66677fig02.jpg" /> 
Figure 2: Disc chip structure diagram. The reaction wells are sequentially numbered counterclockwise, where the inlet/outlet port 1 corresponds to the reaction well number 1. Reaction wells 1, 4, 7, 10, 13, 16, 19, 22, and 24 are negative controls. Reaction well 6 is a positive control (E. coli). The reaction well 12 is an internal positive control, and reaction well 23 is an external positive control. Reaction well 2 detects spn. Reaction well 3 detects sau. The reaction well 5 detects MRSA. The reaction well 8 detects kpn. The reaction well 9 detects pae. The reaction well 11 detects aba. The reaction well 14 detects sma. The reaction well 15 detects hin. Please see Table 5 for sample details. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66677/66677fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
4. Sample preparation and bacterial detection
<ol>
	<li>Prepare the isothermal amplification reaction system
	<ol>
		<li>Take the isothermal amplification reagent from the kit and allow it to thaw at room temperature fully. Gently shake to mix thoroughly and centrifuge briefly to collect at the bottom of the tube.</li>
		<li>In the reagent storage and preparation area, pipette 20 &#181;L of the isothermal amplification reagent into a prepared 200 &#181;L centrifuge tube. Cover the tube and move it to the specimen preparation area with one centrifuge tube per sample.</li>
		<li>In the specimen preparation area, add 34.5 &#181;L of the target nucleic acid sample. Shake gently to mix thoroughly and centrifuge briefly to collect at the bottom of the tube. The total volume of each nucleic acid amplification reaction system is 54.5 &#181;L (Table 3).</li>
	</ol>
	</li>
	<li>Chip loading procedure
	<ol>
		<li>On the chip packaging, label the sample number. Open the microfluidic chip with the packaging label facing up.</li>
		<li>Place the chip with the inlet and outlet ports facing upwards. Using a pipette, draw 50 &#181;L of the prepared amplification reaction system and add it to the main channel of the chip through the inlet port. Stop adding when the main channel is filled, and quickly wipe off any excess liquid around the inlet and outlet ports with lint-free tissue.</li>
		<li>Use tweezers to pick up one sealing film and cover it over the inlet and outlet ports. Press a clean pipette tip onto the sealing film in one direction. Ensure a tight seal.</li>
	</ol>
	</li>
	<li>qPCR reaction
	<ol>
		<li>After the light source of the nucleic acid analyzer has completed preheating, click on the Open Tray button. Place the chip with the front side up on the tray, ensuring that the locating small cylinder emerges from the center gap of the chip to secure it. Align the center positioning device in the tray with the large central hole in the chip.</li>
		<li>Click on the Close Tray button to insert the chip into the nucleic acid analyzer.</li>
		<li>In the Sample Information area on the detection interface, enter the information for testing the sample. Sample number, chip number, and sample type are mandatory; others are optional.</li>
		<li>Click on the Start Detection button in the operation area to initiate the sample detection. The instrument will conduct sample testing according to the preset program. 
		NOTE: After the completion of detection, the accompanying software of the device will automatically conduct data analysis. Simultaneously, the nucleic acid analyzer will initiate an automatic cooling process. Once the temperature drops to 37 &#176;C, the instrument automatically opens the tray and ejects the chip for retrieval. The results will be interpreted automatically by the system (Table 4).</li>
	</ol>
	</li>
	<li>Quality control standards
	<ol>
		<li>One positive quality control and nine negative quality controls are enclosed in each chip in this reagent kit. Ensure that the detection results for the positive quality control is positive, and simultaneously, the results for all nine negative quality controls are negative. 
		NOTE: The results are displayed on the right side of the image stating &#34;Quality Control Results: Experiment Normal (Positive Normal, Negative Normal),&#34; indicating that the test results are valid. Retest is required if any result is incorrect and the sample test result is deemed invalid. One positive internal control using human-specific primers is enclosed in the chip, the result of which is positive for clinical sample testing while being negative if the sample has a low human genomic DNA content, indicating a lower cell count. Recollect and retest samples in such cases.</li>
	</ol>
	</li>
	<li>Results analysis
	<ol>
		<li>After completion of the detection, utilize the second-derivative maximum method combined with commercially protected algorithms in the software to calculate the first inflection point of the S-shaped amplification curve during the rapid amplification phase. Details are described in the &#34;Principles of the Test&#34; part of Respiratory Pathogens Nucleic Acid Detection Kit Instructions (Supplementary File 1). 
		NOTE: The TP value is the time difference between the inflection point and the origin. The result is interpreted based on the TP and positive judgment values. If the TP value for a detection index is less than or equal to the positive judgment value for that index, it is interpreted as positive. If the TP value exceeds the positive judgment value, it is interpreted as negative, according to the criteria for &#34;positive decision value&#34; in the instructions. The &#34;Fluorescence Curve Area&#34; displays the normalized curve, and the &#34;Detection Result&#34; area shows the quality control and detection results for each parameter (Table 5).</li>
	</ol>
	</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Reactant</td>
			<td>Volume (&#181;l)</td>
		</tr>
		<tr>
			<td>Isothermal Amplification Reagent</td>
			<td>20</td>
		</tr>
		<tr>
			<td>Template DNA</td>
			<td>34.5</td>
		</tr>
	</tbody>
</table>
Table 3: Isothermal amplification reaction system.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Step</td>
			<td>One</td>
			<td>Two</td>
		</tr>
		<tr>
			<td>Temperature (&#176;C)</td>
			<td>37</td>
			<td>65</td>
		</tr>
		<tr>
			<td>Time (min)</td>
			<td>3</td>
			<td>47</td>
		</tr>
	</tbody>
</table>
Table 4: Nucleic acid amplification reaction program.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Indicator Name</td>
			<td>Positive Control Value</td>
		</tr>
		<tr>
			<td>Streptococcus pneumoniae (sp)</td>
			<td>30</td>
		</tr>
		<tr>
			<td>Staphylococcus aureus (sau)</td>
			<td>34</td>
		</tr>
		<tr>
			<td>Methicillin-resistant Staphylococcus aureus&#160; (mrsa)</td>
			<td>22</td>
		</tr>
		<tr>
			<td>Klebsiella pneumoniae (kpn)</td>
			<td>29</td>
		</tr>
		<tr>
			<td>Pseudomonas aeruginosa (pae)</td>
			<td>36</td>
		</tr>
		<tr>
			<td>Acinetobacter baumannii (aba)</td>
			<td>36</td>
		</tr>
		<tr>
			<td>Stenotrophomonas maltophilia (sma)</td>
			<td>27</td>
		</tr>
		<tr>
			<td>Haemophilus influenzae (hin)</td>
			<td>36</td>
		</tr>
	</tbody>
</table>
Table 5: Positive control value for infection indicator.

Human Respiratory Bacterial Infection Diagnosis Using Isothermal Amplification

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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. S. B., 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+(LAMP):+A+rapid+and+sensitive+tool+for+quality+assessment+of+meat+products.">Loop-mediated isothermal amplification (LAMP): A rapid and sensitive tool for quality assessment of meat products.</a> Compr Rev Food Sci Food SAF. 16 (6), 1359-1378 (2017).</li><li>Hongling Ou, Y. W., 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=Rapid+detection+of+multiple+pathogens+by+the+combined+loop-mediated+isothermal+amplification+technology+and+microfluidic+chip+technology.">Rapid detection of multiple pathogens by the combined loop-mediated isothermal amplification technology and microfluidic chip technology.</a> Ann Palliat Med. 10 (10), 11053-11066 (2021).</li><li>Liang Wang, J. -. X. L., 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=Quantitative+polymerase+chain+reaction+(qPCR)-based+rapid+diagnosis+of+helicobacter+pylori+infection+and+antibiotic+resistance.">Quantitative polymerase chain reaction (qPCR)-based rapid diagnosis of helicobacter pylori infection and antibiotic resistance.</a> J Vis Exp. (197), e65689 (2023).</li><li>Jing-Wen Lyu, B. X., 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=Rapid+prediction+of+multidrug-resistant+klebsiella+pneumoniae+through+deep+learning+analysis+of+SERS+spectra.">Rapid prediction of multidrug-resistant klebsiella pneumoniae through deep learning analysis of SERS spectra.</a> Microbiol Spectr. 11 (2), e0412622 (2023).</li><li>Wei Liu, J. -. W. T., 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=Discrimination+between+carbapenem-resistant+and+carbapenem-sensitive+klebsiella+pneumoniae+strains+through+computational+analysis+of+surface-enhanced+Raman+spectra+a+pilot+study.">Discrimination between carbapenem-resistant and carbapenem-sensitive klebsiella pneumoniae strains through computational analysis of surface-enhanced Raman spectra a pilot study.</a> Microbiol Spectr. 10 (1), e0240921 (2022).</li><li>Zhang, L. Y., 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=Classification+and+prediction+of+klebsiella+pneumoniae+strains+with+different+MLST+allelic+profiles+via+SERS+spectral+analysis.">Classification and prediction of klebsiella pneumoniae strains with different MLST allelic profiles via SERS spectral analysis.</a> Peer J. 11, e16161 (2023).</li><li>Zhi Liu, Q. Z., 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=Rapid+and+sensitive+detection+of+salmonella+in+chickens+using+loop-mediated+isothermal+amplification+combined+with+a+lateral+flow+dipstick.">Rapid and sensitive detection of salmonella in chickens using loop-mediated isothermal amplification combined with a lateral flow dipstick.</a> J Microbiol Biotechnol. 29 (3), 454-464 (2019).</li><li>Chen, F., 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=Fully+Integrated+microfluidic+platform+for+multiplexed+detection+of+hunov+by+a+dynamic+confined-space-implemented+one-pot+RPA-LAMP+system.">Fully Integrated microfluidic platform for multiplexed detection of hunov by a dynamic confined-space-implemented one-pot RPA-LAMP system.</a> Adv Sci (Weinh). 21, e2306612 (2023).</li></ol>

The authors declare no conflict of interest.

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 precise detection of RTIs. This quick detection system is established by combining LAMP technology with microfluidic technology. The nucleic acid from clinical samples, such as sputum, BLF, and ALF, can be extracted quickly. This extracted material facilitates the rapid and precise detection of bacterial pathogens, guiding clinical practices for improved control and treatment of RTIs.
Previous studies have shown that compared to culture-based methods and polymerase chain reaction, LAMP results achieved 100% sensitivity, specificity, and no cross-reaction with other bacteria in pure cultures and blood samples18. LAMP assays can also detect pathogens directly from biological fluids, such as blood, pleural and peritoneal fluid, and cerebrospinal fluid19. It has been previously demonstrated that exogenous DNA and inhibitors may significantly reduce the sensitivity of PCR, and for LAMP assays, purification of DNA from clinical specimens is not required20. This technique utilizes yeast genes and primers during the amplification process to assess the normalcy of the amplification, thereby determining the qualification of external control quality control. Internal controls, employing primers from human genes, determine the normalcy of sampling, nucleic acid extraction, and amplification processes. Simultaneous utilization of internal and external controls ensures the reliability of the results. At the same time, multiple positive and negative control wells are also set up, which greatly ensures the accuracy of the test. LAMP microfluidic chip has capabilities similar to qPCR in detecting clinical specimens. It has the characteristics of high sensitivity and strong detection ability, providing a reliable basis for rapid clinical detection and precise treatment21. Microbial culture takes 3-5 days or even longer, and some pathogens, such as Chlamydia pneumoniae and Legionella pneumophila, cannot be cultured. In contrast, LAMP detects multiple bacterial pathogens, even the unculturable, in less than 3 h, significantly increasing detection speed.
It can also perform multiple detections. The LAMP microfluidic chip is divided into various reaction units that detect multiple pathogens in parallel, ensuring the independence of each reaction and the accuracy of the results22,23. The data from the China Network Antibacterial Surveillance Center showed that the prevalence of K. pneumoniae ranked first among the bacterial isolates from respiratory specimens in 202215. Among the respiratory samples collected by Guangdong Provincial People&#39;s Hospital in 2022, the positive rate of K. pneumoniae tested by LAMP was 18.6%, which is also one of the bacteria with the highest prevalence. In all K. pneumoniae-positive samples, 30.6% were infections caused solely by K. pneumoniae, and 29.7% were infections where K. pneumoniae coexisted with other bacterial species. Additionally, there were triple infections (23.9%) and quadruple infections (10.5%) involving K. pneumoniae infection, constituting the majority in KP-positive samples, approximately 95%. There were very few cases of quintuple and sextuple infections, and as of now, infections beyond sextuple have not been detected. The results demonstrate six representative scenarios, each representing the detection results of infections caused by K. pneumoniae alone or coexisting with 1-5 other bacterial species.
In addition, the LAMP chip technology used in this study can also detect bacterial resistance. It detects methicillin-resistant MRSA via the mecA gene. When the wells representing Staphylococcus aureus and the wells for the mecA gene are positive, they represent a methicillin-MRSA infection. Researchers are continuously exploring methods for simultaneously rapidly detecting pathogens and antibiotic resistance traits. For example, a rapid diagnostic method for Helicobacter pylori infection and antibiotic resistance is based on quantitative polymerase chain reaction (qPCR)24. Another approach involves quickly predicting multidrug-resistant K. pneumoniae through deep learning analysis of surface-enhanced Raman scattering (SERS) spectra25 and a method for detecting K. pneumoniae and carbapenemase genes using SERS spectra26. Additionally, there is a classification and prediction method for different multi-locus sequence typing (MLST) profiles of K. pneumoniae strains based on SERS spectra analysis27. We will also subsequently develop sensitive and accurate detection of K. pneumoniae and carbapenemase genes, with performance comparable to gold-standard clinical methods that are time-consuming or based on expensive specialized instruments. Moreover, the ideal diagnostic approach is fast, accurate, cost-effective, and user-friendly18. A study estimating the cost of reagents for current diagnostic technologies showed that sample testing based on PCR technology costs up to US$6.4-7.7, compared with only US$0.71-2 for LAMP technology28.
The multi-channel loop-mediated isothermal amplification (LAMP) method, while offering significant advantages for the rapid detection of bacterial pathogens in respiratory tract infections, has its limitations. The high sensitivity of LAMP poses a risk of cross-contamination, where minimal DNA contamination can lead to false-positive results29; thus, stringent sample handling and procedural protocols are necessary to mitigate this risk. Despite the method&#39;s simplicity, the interpretation of results requires specific expertise and experience to avoid misinterpretation or diagnostic errors, emphasizing the need for well-trained personnel. Recognizing these limitations is essential for the effective application and further development of the LAMP method in the field of clinical diagnostics. Taken together, the LAMP method combined with a microfluidic chip can rapidly detect various bacterial pathogens in parallel. It has the advantages of high sensitivity and strong specificity and the features of low cost, rapidness, and convenience. It shows considerable practical significance for preventing and controlling RTIs caused by pathogenic infections in clinical settings. The significant feature of the LAMP test proposed in this study is that it is easy for laboratory staff to master, which is of great help to primary hospitals, disease prevention, control centers, and inspection and quarantine departments. In summary, the LAMP microfluidic chip system is a powerful detection tool in clinical laboratories, which can solve the high expenditure and low turn-around-time challenges in clinical infection diagnosis and has significant application values in clinical settings.

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.remotexs.ntu.edu.sg/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.

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. This one-step process completes the amplification and detection of the target gene. This study selected 7 samples with different experimental outcomes to demonstrate the reliability of the experimental design. The capital letter S stands for a sample. S1 is a sample infected with a single strain of K. pneumoniae. S2 to S7 are samples with polymicrobial infections, all infected with K. pneumoniae. For effective detection results, interpretation can be made through the normalized fluorescence curves displayed in the images. A positive indicator is represented by an amplification curve exhibiting an S-shape, while a horizontal line represents a negative indicator (Figure 3).
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66677/66677fig03.jpg" /> 
Figure 3: Representative nucleic acid amplification results via LAMP. The capital letter S stands for sample. S1 is a sample infected with a single strain of kpn. S2 to S7 are samples with polymicrobial infections, all infected with kpn. S2 represents a sample infected with kpn and sma. S3 denotes a sample concurrently infected with kpn, sau, and MRSA. S4 signifies a sample concurrently infected with kpn, sau, MRSA, and aba. S5 indicates a sample concurrently infected with kpn, sma, sau, MRSA, and aba. S6 denotes a sample concurrently infected with kpn, sma, pae, sau, MRSA, and aba. S7 represents a sample concurrently infected with kpn, sma, pae, sau, MRSA, aba, and E. coli. Please see Table 5 for sample details. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66677/66677fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
This reagent kit utilizes the ROC method to calculate positive threshold values for eight test indicators. When the TP value of a detection indicator is less than or equal to its corresponding positive threshold value, it is interpreted as positive. Conversely, when the TP value of a detection indicator is greater than its positive threshold value, it is interpreted as negative. The positive threshold values for each indicator are detailed in Table 5. The true positive TP values we detected for the 7 samples with different experimental outcomes are shown in Table 6.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td rowspan="2">SAMPLE</td>
			<td>kpn</td>
			<td>sma</td>
			<td>pae</td>
			<td>sau</td>
			<td>mrsa</td>
			<td>hin</td>
			<td>aba</td>
			<td>eco</td>
		</tr>
		<tr>
			<td>TP (+/-)</td>
			<td>TP (+/-)</td>
			<td>TP (+/-)</td>
			<td>TP (+/-)</td>
			<td>TP (+/-)</td>
			<td>TP (+/-)</td>
			<td>TP (+/-)</td>
			<td>TP (+/-)</td>
		</tr>
		<tr>
			<td>S1</td>
			<td>19.68 (+)</td>
			<td>U (-)</td>
			<td>U (-)</td>
			<td>U (-)</td>
			<td>U (-)</td>
			<td>U (-)</td>
			<td>U (-)</td>
			<td>U (-)</td>
		</tr>
		<tr>
			<td>S2</td>
			<td>17.73 (+)</td>
			<td>18.30 (+)</td>
			<td>U (-)</td>
			<td>U (-)</td>
			<td>U (-)</td>
			<td>U (-)</td>
			<td>U (-)</td>
			<td>U (-)</td>
		</tr>
		<tr>
			<td>S3</td>
			<td>16.73 (+)</td>
			<td>U (-)</td>
			<td>U (-)</td>
			<td>18.05 (+)</td>
			<td>12.18 (+)</td>
			<td>U (-)</td>
			<td>U (-)</td>
			<td>U (-)</td>
		</tr>
		<tr>
			<td>S4</td>
			<td>21.66 (+)</td>
			<td>U (-)</td>
			<td>U (-)</td>
			<td>27.07 (+)</td>
			<td>19.61 (+)</td>
			<td>U (-)</td>
			<td>18.29 (+)</td>
			<td>U (-)</td>
		</tr>
		<tr>
			<td>S5</td>
			<td>16.16 (+)</td>
			<td>20.85 (+)</td>
			<td>U (-)</td>
			<td>23.80 (+)</td>
			<td>17.13 (+)</td>
			<td>U (-)</td>
			<td>15.22 (+)</td>
			<td>U (-)</td>
		</tr>
		<tr>
			<td>S6</td>
			<td>11.20 (+)</td>
			<td>16.79(+)</td>
			<td>17.18(+)</td>
			<td>23.09 (+)</td>
			<td>16.14 (+)</td>
			<td>U (-)</td>
			<td>14.28 (+)</td>
			<td>U (-)</td>
		</tr>
		<tr>
			<td>S7</td>
			<td>14.69 (+)</td>
			<td>24.84(+)</td>
			<td>19.70(+)</td>
			<td>24.07(+)</td>
			<td>14.16 (+)</td>
			<td>- (+)</td>
			<td>15.23 (+)</td>
			<td>20.12 (+)</td>
		</tr>
		<tr>
			<td colspan="9">Each Sample+/-, qualitative result; 
			&#160;+, positive result; -, negative result.&#160;</td>
		</tr>
	</tbody>
</table>
Table 6: Table of TP values for detection after infection.
Supplementary Table 1: List of abbreviations. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66677/Supplementary table.zip" target="_blank">Please click here to download this File.</a>
Supplementary File 1: Respiratory pathogens nucleic acid detection kit instructions. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66677/Supplementary file. Kit Instructions.zip" target="_blank">Please click here to download this File.</a>

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

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

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Video: Auteur in de schijnwerpers: Bevordering van snelle detectie van respiratoire pathogenen met behulp van microfluïdische chip 

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in situ hybridization (FISH) for rapid culture-independent detection of Salmonella spp. Cell-charged tapes can also be placed face-down on selective agar for solid-phase enrichment prior to detection. Alternatively, low-volume liquid enrichments (liquid surface miniculture) can be performed on the surface of the tape in non-selective broth, followed by FISH and analysis via flow cytometry. To begin, sterile adhesive tape is brought into contact with fresh produce, gentle pressure is applied, and the tape is removed, physically extracting microbes present on these surfaces. Tapes are mounted sticky-side up onto glass microscope slides and the sampled cells are fixed with 10% formalin (30 min) and dehydrated using a graded ethanol series (50, 80, and 95%; 3 min each concentration). Next, cell-charged tapes are spotted with buffer containing a Salmonella-targeted DNA probe cocktail and hybridized for 15 - 30 min at 55&deg;C, followed by a brief rinse in a washing buffer to remove unbound probe. Adherent, FISH-labeled cells are then counterstained with the DNA dye 4',6-diamidino-2-phenylindole (DAPI) and results are viewed using fluorescence microscopy. For solid-phase enrichment, cell-charged tapes are placed face-down on a suitable selective agar surface and incubated to allow in situ growth of Salmonella microcolonies, followed by FISH and microscopy as described above. For liquid surface miniculture, cell-charged tapes are placed sticky side up and a silicone perfusion chamber is applied so that the tape and microscope slide form the bottom of a water-tight chamber into which a small volume (&le; 500 &mu;L) of Trypticase Soy Broth (TSB) is introduced. The inlet ports are sealed and the chambers are incubated at 35 - 37&deg;C, allowing growth-based amplification of tape-extracted microbes. Following incubation, inlet ports are unsealed, cells are detached and mixed with vigorous back and forth pipetting, harvested via centrifugation and fixed in 10% neutral buffered formalin. Finally, samples are hybridized and examined via flow cytometry to reveal the presence of Salmonella spp. As described here, our "tape-FISH" approach can provide simple and rapid sampling and detection of Salmonella on tomato surfaces. We have also used this approach for sampling other types of fresh produce, including spinach and jalape&ntilde;o peppers.

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple adhesive-tape-based approach for sampling of tomato and other fresh produce surfaces, followed by rapid whole cell detection of Salmonella using fluorescence in situ hybridization (FISH).

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in ...

Using Luciferase to Image Bacterial Infections in Mice

Imaging is a valuable technique that can be used to monitor biological processes. In particular, the presence of cancer cells, stem cells, specific immune cell types, viral pathogens, parasites and bacteria can be followed in real-time within living animals 1-2. Application of bioluminescence imaging to the study of pathogens has advantages as compared to conventional strategies for analysis of infections in animal models3-4. Infections can be visualized within individual animals over time, without requiring euthanasia to determine the location and quantity of the pathogen. Optical imaging allows comprehensive examination of all tissues and organs, rather than sampling of sites previously known to be infected. In addition, the accuracy of inoculation into specific tissues can be directly determined prior to carrying forward animals that were unsuccessfully inoculated throughout the entire experiment. Variability between animals can be controlled for, since imaging allows each animal to be followed individually. Imaging has the potential to greatly reduce animal numbers needed because of the ability to obtain data from numerous time points without having to sample tissues to determine pathogen load3-4.
This protocol describes methods to visualize infections in live animals using bioluminescence imaging for recombinant strains of bacteria expressing luciferase. The click beetle (CBRLuc) and firefly luciferases (FFluc) utilize luciferin as a substrate5-6. The light produced by both CBRluc and FFluc has a broad wavelength from 500 nm to 700 nm, making these luciferases excellent reporters for the optical imaging in living animal models7-9. This is primarily because wavelengths of light greater than 600 nm are required to avoid absorption by hemoglobin and, thus, travel through mammalian tissue efficiently. Luciferase is genetically introduced into the bacteria to produce light signal10. Mice are pulmonary inoculated with bioluminescent bacteria intratracheally to allow monitoring of infections in real time. After luciferin injection, images are acquired using the IVIS Imaging System. During imaging, mice are anesthetized with isoflurane using an XGI-8 Gas Anethesia System. Images can be analyzed to localize and quantify the signal source, which represents the bacterial infection site(s) and number, respectively. After imaging, CFU determination is carried out on homogenized tissue to confirm the presence of bacteria. Several doses of bacteria are used to correlate bacterial numbers with luminescence. Imaging can be applied to study of pathogenesis and evaluation of the efficacy of antibacterial compounds and vaccines.

Methods for bioluminescence imaging of bacterial infections in living animals are decribed. Pathogens are modified to express luciferase allowing optical whole body imaging of infections in live animals. Animal models can be infected with luciferase expressing pathogens and the resulting course of disease visualized in real-time by bioluminescence imaging.

Imaging is a valuable technique that can be used to monitor biological processes.  In particular, the presence of cancer cells, stem cells, specific ...

'Bioluminescent' Reporter Phage for the Detection of Category A Bacterial Pathogens

Yersinia pestis and Bacillus anthracis are Category A bacterial pathogens that are the causative agents of the plague and anthrax, respectively 1. Although the natural occurrence of both diseases&#39; is now relatively rare, the possibility of terrorist groups using these pathogens as a bioweapon is real. Because of the disease&#39;s inherent communicability, rapid clinical course, and high mortality rate, it is critical that an outbreak be detected quickly. Therefore methodologies that provide rapid detection and diagnosis are essential to ensure immediate implementation of public health measures and activation of crisis management.
Recombinant reporter phage may provide a rapid and specific approach for the detection of Y. pestis and B. anthracis. The Centers for Disease Control and Prevention currently use the classical phage lysis assays for the confirmed identification of these bacterial pathogens 2-4. These assays take advantage of naturally occurring phage which are specific and lytic for their bacterial hosts. After overnight growth of the cultivated bacterium in the presence of the specific phage, the formation of plaques (bacterial lysis) provides a positive identification of the bacterial target. Although these assays are robust, they suffer from three shortcomings: 1) they are laboratory based; 2) they require bacterial isolation and cultivation from the suspected sample, and 3) they take 24-36 h to complete. To address these issues, recombinant &#34;light-tagged&#34; reporter phage were genetically engineered by integrating the Vibrio harveyi luxAB genes into the genome of Y. pestis and B. anthracis specific phage 5-8. The resulting luxAB reporter phage were able to detect their specific target by rapidly (within minutes) and sensitively conferring a bioluminescent phenotype to recipient cells. Importantly, detection was obtained either with cultivated recipient cells or with mock-infected clinical specimens 7.
For demonstration purposes, here we describe the method for the phage-mediated detection of a known Y. pestis isolate using a luxAB reporter phage constructed from the CDC plague diagnostic phage &#934;A1122 6,7 (Figure 1). A similar method, with minor modifications (e.g. change in growth temperature and media), may be used for the detection of B. anthracis isolates using the B. anthracis reporter phage W&#946;::luxAB 8. The method describes the phage-mediated transduction of a biolumescent phenotype to cultivated Y. pestis cells which are subsequently measured using a microplate luminometer. The major advantages of this method over the traditional phage lysis assays is the ease of use, the rapid results, and the ability to test multiple samples simultaneously in a 96-well microtiter plate format.
<img alt="Figure 1" src="/files/ftp_upload/2740/2740fig1.jpg" /> 
Figure 1. Detection schematic. The phage are mixed with the sample, the phage infects the cell, luxAB are expressed, and the cell bioluminesces. Sample processing is not necessary; the phage and cells are mixed and subsequently measured for light.

A simple method for the identification of priority bacterial pathogens is to use genetically engineered reporter phage. These reporter phage, which are specific to their particular host species, are capable of rapidly transducing a bioluminescent signal response to host cells. Herein, we describe the use of reporter phage for the detection of Yersinia pestis.

Yersinia pestis and Bacillus anthracis are Category A bacterial pathogens that are the causative agents of the plague and anthrax, respectively 1. ...

Infection of Zebrafish Embryos with Intracellular Bacterial Pathogens

Zebrafish (Danio rerio) embryos are increasingly used as a model for studying the function of the vertebrate innate immune system in host-pathogen interactions 1. The major cell types of the innate immune system, macrophages and neutrophils, develop during the first days of embryogenesis prior to the maturation of lymphocytes that are required for adaptive immune responses. The ease of obtaining large numbers of embryos, their accessibility due to external development, the optical transparency of embryonic and larval stages, a wide range of genetic tools, extensive mutant resources and collections of transgenic reporter lines, all add to the versatility of the zebrafish model. Salmonella enterica serovar Typhimurium (S. typhimurium) and Mycobacterium marinum can reside intracellularly in macrophages and are frequently used to study host-pathogen interactions in zebrafish embryos. The infection processes of these two bacterial pathogens are interesting to compare because S. typhimurium infection is acute and lethal within one day, whereas M. marinum infection is chronic and can be imaged up to the larval stage 2, 3. The site of micro-injection of bacteria into the embryo (Figure 1) determines whether the infection will rapidly become systemic or will initially remain localized. A rapid systemic infection can be established by micro-injecting bacteria directly into the blood circulation via the caudal vein at the posterior blood island or via the Duct of Cuvier, a wide circulation channel on the yolk sac connecting the heart to the trunk vasculature. At 1 dpf, when embryos at this stage have phagocytically active macrophages but neutrophils have not yet matured, injecting into the blood island is preferred. For injections at 2-3 dpf, when embryos also have developed functional (myeloperoxidase-producing) neutrophils, the Duct of Cuvier is preferred as the injection site. To study directed migration of myeloid cells towards local infections, bacteria can be injected into the tail muscle, otic vesicle, or hindbrain ventricle 4-6. In addition, the notochord, a structure that appears to be normally inaccessible to myeloid cells, is highly susceptible to local infection 7. A useful alternative for high-throughput applications is the injection of bacteria into the yolk of embryos within the first hours after fertilization 8. Combining fluorescent bacteria and transgenic zebrafish lines with fluorescent macrophages or neutrophils creates ideal circumstances for multi-color imaging of host-pathogen interactions. This video article will describe detailed protocols for intravenous and local infection of zebrafish embryos with S. typhimurium or M. marinum bacteria and for subsequent fluorescence imaging of the interaction with cells of the innate immune system.

Transparent zebrafish embryos have proved useful model hosts to visualize and functionally study interactions between innate immune cells and intracellular bacterial pathogens, such as Salmonella typhimurium and Mycobacterium marinum. Micro-injection of bacteria and multi-color fluorescence imaging are essential techniques involved in the application of zebrafish embryo infection models.

Zebrafish (Danio rerio) embryos are increasingly used as a model for studying the function of the vertebrate innate immune system in host-pathogen ...

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The apical and basolateral surfaces of airway epithelial cells demonstrate directional responses to pathogen exposure in vivo. Thus, ideal in vitro models for examining cellular responses to respiratory pathogens polarize, forming apical and basolateral surfaces. One such model is differentiated normal human bronchial epithelial cells (NHBE). However, this system requires lung tissue samples, expertise isolating and culturing epithelial cells from tissue, and time to generate an air-liquid interface culture.
Calu-3 cells, derived from a human bronchial adenocarcinoma, are an alternative model for examining the response of proximal airway epithelial cells to respiratory insult1, pharmacological compounds2-6, and bacterial7-9 and viral pathogens, including influenza virus, rhinovirus and severe acute respiratory syndrome - associated coronavirus10-14. Recently, we demonstrated that Calu-3 cells are susceptible to respiratory syncytial virus (RSV) infection in a manner consistent with NHBE15,16 . Here, we detail the establishment of a polarized, liquid-covered culture (LCC) of Calu-3 cells, focusing on the technical details of growing and culturing Calu-3 cells, maintaining cells that have been cultured into LCC, and we present the method for performing respiratory virus infection of polarized Calu-3 cells.
To consistently obtain polarized Calu-3 LCC, Calu-3 cells must be carefully subcultured before culturing in Transwell inserts. Calu-3 monolayer cultures should remain below 90% confluence, should be subcultured fewer than 10 times from frozen stock, and should regularly be supplied with fresh medium. Once cultured in Transwells, Calu-3 LCC must be handled with care. Irregular media changes and mechanical or physical disruption of the cell layers or plates negatively impact polarization for several hours or days. Polarization is monitored by evaluating trans-epithelial electrical resistance (TEER) and is verified by evaluating the passive equilibration of sodium fluorescein between the apical and basolateral compartments17,18 . Once TEER plateaus at or above 1,000 &Omega;&times;cm2, Calu-3 LCC are ready to use to examine cellular responses to respiratory pathogens.

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention.

The  apical and basolateral surfaces of airway epithelial cells demonstrate  directional responses to pathogen exposure in  vivo. Thus, ideal in vitro ...

Loop-mediated Isothermal Amplification (LAMP) Assays for the Species-specific Detection of Eimeria that Infect Chickens

Eimeria species parasites, protozoa which cause the enteric disease coccidiosis, pose a serious threat to the production and welfare of chickens. In the absence of effective control clinical coccidiosis can be devastating. Resistance to the chemoprophylactics frequently used to control Eimeria is common and sub-clinical infection is widespread, influencing feed conversion ratios and susceptibility to other pathogens such as Clostridium perfringens. Despite the availability of polymerase chain reaction (PCR)-based tools, diagnosis of Eimeria infection still relies almost entirely on traditional approaches such as lesion scoring and oocyst morphology, but neither is straightforward. Limitations of the existing molecular tools include the requirement for specialist equipment and difficulties accessing DNA as template. In response a simple field DNA preparation protocol and a panel of species-specific loop-mediated isothermal amplification (LAMP) assays have been developed for the seven Eimeria recognised to infect the chicken. We now provide a detailed protocol describing the preparation of genomic DNA from intestinal tissue collected post-mortem, followed by setup and readout of the LAMP assays. Eimeria species-specific LAMP can be used to monitor parasite occurrence, assessing the efficacy of a farm&#8217;s anticoccidial strategy, and to diagnose sub-clinical infection or clinical disease with particular value when expert surveillance is unavailable.

Loop-mediated Isothermal Amplification LAMP Assays for the Species-specific Detection of Eimeria that Infect Chickens

Diagnosis of Eimeria infection in chickens remains demanding. Parasite morphology- and host pathology-led approaches are commonly inconclusive, while molecular approaches based on PCR have proven demanding in cost and expertise. The aim of this protocol is to establish loop-mediated isothermal amplification (LAMP) as a straightforward molecular diagnostic for eimerian infection.

Eimeria species parasites, protozoa which cause the enteric disease coccidiosis, pose a serious threat to the production and welfare of chickens. In the ...

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence factors, which can subvert and manipulate key host functions. The study of host/pathogen interactions is therefore extremely important to understand bacterial infections and develop alternative strategies to counter infectious diseases. This approach however, requires the development of new high-throughput assays for the unbiased, automated identification and characterization of bacterial virulence determinants. Here, we describe a method for the generation of a GFP-tagged mutant library by transposon mutagenesis and the development of high-content screening approaches for the simultaneous identification of multiple transposon-associated phenotypes. Our working model is the intracellular bacterial pathogen Coxiellaburnetii, the etiological agent of the zoonosis Q fever, which is associated with severe outbreaks with a consequent health and economic burden. The obligate intracellular nature of this pathogen has, until recently, severely hampered the identification of bacterial factors involved in host pathogen interactions, making of Coxiella the ideal model for the implementation of high-throughput/high-content approaches.

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Coxiella burnetii is an obligate intracellular Gram-negative bacterium responsible for the zoonotic disease Q fever. Here we describe methods for the generation of Coxiella fluorescent transposon mutants as well as the automated identification and analysis of the resulting internalization, replication and cytotoxic phenotypes.

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence ...

The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii

Coxiella burnetii, the agent causing Q fever, is an obligate intracellular bacterium. PCR based diagnostic assays have been developed for detecting C. burnetii DNA in cell cultures and clinical samples. PCR requires specialized equipment and extensive end user training, and therefore, it is not suitable for routine work especially in a resource-constrained area. We have developed a loop-mediated isothermal amplification (LAMP) assay to detect the presence of C. burnetii in patient samples. This method is performed at a single temperature around 60 &#176;C in a water bath or heating block. The sensitivity of this LAMP assay is very similar to PCR with a detection limit of about 25 copies per reaction. This report describes the preparation of the reaction using lyophilized reagents and visualization of results using hydroxynaphthol blue (HNB) or a UV lamp with fluorescent intercalating dye in the reaction. The LAMP reagents were lyophilized and stored at room temperature (RT) for one month without loss of detection sensitivity. This LAMP assay is particularly robust because the reaction mixture preparation does not involve complex steps. This method is ideal for use in resource-limited settings where Q fever is endemic.

The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii

We described here a simple loop-mediated isothermal amplification (LAMP) method using lyophilized reagents for the detection of C. burnetii in patient samples.

Coxiella burnetii, the agent causing Q fever, is an obligate intracellular bacterium. PCR based diagnostic assays have been developed for detecting C. ...

High-throughput Detection of Respiratory Pathogens in Animal Specimens by Nanoscale PCR

Nanoliter scale real-time PCR uses spatial multiplexing to allow multiple assays to be run in parallel on a single plate without the typical drawbacks of combining reactions together. We designed and evaluated a panel based on this principle to rapidly identify the presence of common disease agents in dogs and horses with acute respiratory illness. This manuscript describes a nanoscale diagnostic PCR workflow for sample preparation, amplification, and analysis of target pathogen sequences, focusing on procedures that are different from microliter scale reactions. In the respiratory panel presented, 18 assays were each set up in triplicate, accommodating up to 48 samples per plate. A universal extraction and pre-amplification workflow was optimized for high-throughput sample preparation to accommodate multiple matrices and DNA and RNA based pathogens. Representative data are presented for one RNA target (influenza A matrix) and one DNA target (equine herpesvirus 1). The ability to quickly and accurately test for a comprehensive, syndrome-based group of pathogens is a valuable tool for improving efficiency and ergonomics of diagnostic testing and for acute respiratory disease diagnosis and management.

High-throughput testing of DNA and RNA based pathogens by nanoscale PCR is described using a syndromic canine and equine respiratory PCR panel.

Nanoliter scale real-time PCR uses spatial multiplexing to allow multiple assays to be run in parallel on a single plate without the typical drawbacks of ...

A High-throughput Shigella-specific Bactericidal Assay

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing activity by assessing the ability of antibodies in serum to bind to bacteria and activate complement. This complement activation results in the lysis and killing of the target bacteria. These assays are valuable because they go beyond quantifying antibody production to elucidate the biological functions that these antibodies have, allowing researchers to study the role that antibodies may play in preventing infection. SBAs have been used to study immune responses for many human pathogens, but there is no widely accepted methodology for Shigella at present. Historically, SBAs have been very labor-intensive, requiring many time-consuming steps to accurately quantify surviving bacteria. This protocol describes a simple, robust, and high-throughput assay that measures functional antibodies specific for Shigella in serum in vitro. The method described here offers many advantages over traditional SBAs, including the use of frozen bacterial stocks, 96 well assay plates, a micro-culture system, and automated colony-counting. All of these modifications make this assay less labor-intensive and more high-throughput. This protocol is simpler and faster to perform than traditional SBAs while still using simple technologies and readily available reagents. The protocol has been successfully applied in multiple independent laboratories and the assay is robust and reproducible. The assay can be used to assess immune responses in pre-clinical as well as clinical studies. Quantifying shigellacidal antibody titers both before and after antigen exposure (either by immunization or infection) allows for a broader understanding of how functional antibody responses are generated and their contribution to protective immunity. The development of this standardized, well-characterized assay may greatly facilitate Shigella vaccine design.

A High-throughput Shigella-specific Bactericidal Assay

Here we present a protocol to measure Shigellacidal activity of antibodies in serum. Serum is mixed with bacteria and exogenous complement, incubated, and the reaction mixture is plated on agar plates. Viable bacteria form colonies which are counted, using an automated colony enumerator, and used to determine the bactericidal titer.

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing ...

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

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