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'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'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.

Figure 1: Schematic illustration of the LAMP detection method. Please click here to view a larger version of this figure.

Extraction of DNA from Human Sputum Samples for the Rapid Detection of Bacterial Pathogens Using a Microfluidic Chip