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

This study focused on the rapid identification of bacterial pathogens causing respiratory tract infections by microfluidic chip technology based on loop-mediated isothermal amplification and the effectiveness of this approach in the management and control respiratory infections in the hospital and communities. The main challenge of this method is the risk of socialities with its high sensitivity, which necessitates strict sample handling and the standard operational protocols to reduce the risk of false-positive results. Additionally, laboratory personnel needed to master the relevant skills to effectively apply this method in primary hospitals.

Compared to traditional PCR techniques, The microfluidic chip technology based on loop-mediated isothermal amplification has high specificity and the sensitivity. It can quickly detect up to certain high frequency respiratory pathogens and common resistance genes within one novel. The technique we demonstrated in this study has important practical implications for the control and treatment of respiratory infections, helping to prevent the spread of bacterial pathogens and providing guidance for the appropriate antibiotic treatment.

We intend to continue developing novel diagnostic measures with a focus on rapid and accurate detection of microbial pathogens causing infection diseases. We'll also endeavor to work on developing novel point care testing methods for infectious diseases that can be easily deployed in resource-limited settings like rural China. To begin cleanse the oral cavity and teeth with clean water.

Ask the patients with dentures to remove them. Instruct the patient to cough forcefully to expel deep respiratory sputum into a sputum container. To extract the DNA, add an appropriate volume of 10%sodium hydroxide to the sample based on its viscosity.

Vortex the suspension for 15 seconds. Then incubate it at 37 degrees Celsius for 30 minutes to liquefy the sample. Next, pipette one milliliter of the liquified sample into a 1.5 milliliter centrifuge tube.

Centrifuge the sample at 15, 777 G for five minutes at two to six degrees Celsius. Then, pipette out the supernatant to discard it. Now, add one milliliter of the washing solution to the tube, and vortex it to lift the precipitate from the tube's bottom.

After centrifuging and discarding the supernatant as before, pipette 100 microliters of nucleic acid extraction solution to the pellet. With a pipette, aspirate and thoroughly mix the precipitate with the solution. Transfer the liquid and precipitate together into a nucleic acid extraction tube.

Place the tube in a vortex mixer at medium speed for five minutes. Then, transfer the tube to a metal bath at a constant temperature of 100 degrees Celsius for five minutes. Finally, centrifuge the samples at 15, 777 G for five minutes at two to six degrees Celsius before further experimentation.

To begin, gently shake a thawed isothermal amplification reagent. Centrifuge the reagent to collect all of it at the bottom of the tube. Pipette 20 microliters of the isothermal amplification reagent into a prepared 200 microliter centrifuge tube.

Cover the tube with its lid and transfer it to the specimen preparation area. Now add 34.5 microliters of the target nucleic acid sample into each tube. Shake gently to mix thoroughly.

Centrifuge the tubes briefly to collect all samples at the bottom of the tubes. Next, write the sample number on a chip-packaging label. Open the microfluidic chip with the packaging label facing up.

Place the chip with the inlet and outlet ports facing upwards. Then use a pipette to draw 50 microliters 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.

Wipe away any excess liquid around the inlet and outlet ports with lint-free tissue. Next, click on the open tray button on the nucleic acid analyzer after it is preheated. 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.

Press the closed tray button to insert the chip into the nucleic acid analyzer. Enter the sample testing information in the sample information area on the detection interface. Then, press the start detection button to initiate sample detection.

Sample one was found to be infected with Klebsiella pneumonia, while samples two and seven showed polymicrobial infections. A positive indicator was represented by an amplification curve exhibiting an S shape, while a horizontal line represented a negative indicator.