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

All samples for this study were evaluated and approved by the Ethics Review Committee of Guangdong Provincial People'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

1. Sputum collection
   1. 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).
   2. Avoid contamination with saliva or nasal discharge. If coughing is difficult, administer inhalation of a 100 g/L NaCl solution at 45 °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.
2. Bronchial lavage fluid (BLF)
   1. 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.
   2. 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.
3. Alveolar lavage fluid (ALF)
   1. Identify the lesion location through chest imaging examinations. Select the most significant or rapidly progressing area for lavage.
   2. 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.
   3. 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.
   4. Administer 30-50 mL saline per session, with 100-250 mL volume (max. 300 mL). Collect the extracted mucus in a sealed sterile container.

2. DNA extraction

1. 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.
   1. 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 °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.
2. Pipette 1 mL of the liquefied sample into a 1.5 mL centrifuge tube. Centrifuge at a speed of 15,777 × g for 5 min at 2-6 °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 × g for 1 min before aspiration.
3. 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.
   1. Centrifuge the solution at 15,777 × 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.
4. Add 100 µL 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.
5. 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 °C for 5 min.
6. Centrifuge at 15,777 × g at 2-6 °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 °C. After the amplification, store the nucleic acid at -20 °C. For long-term storage (beyond 24 h), store the nucleic acid at -20 °C. When ready to use again, thaw the sample naturally, vortex to mix, heat in a 95 °C water bath for 5 min, centrifuge at 10,956 × g for 1 min, and use the supernatant for PCR amplification.

Table 1: Composition of nucleic acid extraction reagent.

3. Loop-mediated isothermal amplification and microfluid chip

1. Reagent and microfluidic chip
   1. Conduct isothermal amplification reactions on a microfluidic disk-shaped chip (see Table of Materials). Carry out the constant-temperature amplification at 65 °C.
   2. 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.
2. Loop-mediated isothermal amplification
   1. 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).
   2. 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 °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.
3. 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.
   1. 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 × 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.

Table 2: Composition of nucleic acid detection kit for respiratory tract pathogens.

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. Please click here to view a larger version of this figure.

4. Sample preparation and bacterial detection

1. Prepare the isothermal amplification reaction system
   1. 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.
   2. In the reagent storage and preparation area, pipette 20 µL of the isothermal amplification reagent into a prepared 200 µL centrifuge tube. Cover the tube and move it to the specimen preparation area with one centrifuge tube per sample.
   3. In the specimen preparation area, add 34.5 µL 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 µL (Table 3).
2. Chip loading procedure
   1. On the chip packaging, label the sample number. Open the microfluidic chip with the packaging label facing up.
   2. Place the chip with the inlet and outlet ports facing upwards. Using a pipette, draw 50 µL 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.
   3. 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.
3. qPCR reaction
   1. 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.
   2. Click on the Close Tray button to insert the chip into the nucleic acid analyzer.
   3. 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.
   4. 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 °C, the instrument automatically opens the tray and ejects the chip for retrieval. The results will be interpreted automatically by the system (Table 4).
4. Quality control standards
   1. 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 "Quality Control Results: Experiment Normal (Positive Normal, Negative Normal)," 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.
5. Results analysis
   1. 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 "Principles of the Test" 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 "positive decision value" in the instructions. The "Fluorescence Curve Area" displays the normalized curve, and the "Detection Result" area shows the quality control and detection results for each parameter (Table 5).

Table 3: Isothermal amplification reaction system.

Table 4: Nucleic acid amplification reaction program.

Table 5: Positive control value for infection indicator.