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 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 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.
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.
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
2. DNA extraction
Solution | Components | Number | Specification |
Washing Solution | 10 mM EDTA | 1 bottle | 24 mL/bottle |
Nucleic Acid Extraction Reagent | 10mM Tris-HCl, 1mM EDTA, Nucleic acid preservatives | 2 tubes | 1.2 mL/bottle |
Nucleic Acid Extraction Tube | Glass beads | 1 bag | 24 bottles/bag |
Table 1: Composition of nucleic acid extraction reagent.
3. Loop-mediated isothermal amplification and microfluid chip
Components | Composition | Number |
Chip | Primers | 12 places |
Sealing Film | / | 1 sheet |
Isothermal Amplification Reagent | Fluorescent dye, Enzyme | 270 µL/tube |
Positive Control | Escherichia coli Genomic DNA | 160 µL/tube |
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
Reactant | Volume (µl) |
Isothermal Amplification Reagent | 20 |
Template DNA | 34.5 |
Table 3: Isothermal amplification reaction system.
Step | One | Two |
Temperature (°C) | 37 | 65 |
Time (min) | 3 | 47 |
Table 4: Nucleic acid amplification reaction program.
Indicator Name | Positive Control Value |
Streptococcus pneumoniae (sp) | 30 |
Staphylococcus aureus (sau) | 34 |
Methicillin-resistant Staphylococcus aureus (mrsa) | 22 |
Klebsiella pneumoniae (kpn) | 29 |
Pseudomonas aeruginosa (pae) | 36 |
Acinetobacter baumannii (aba) | 36 |
Stenotrophomonas maltophilia (sma) | 27 |
Haemophilus influenzae (hin) | 36 |
Table 5: Positive control value for infection indicator.
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 °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).
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. Please click here to view a larger version of this figure.
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.
SAMPLE | kpn | sma | pae | sau | mrsa | hin | aba | eco |
TP (+/-) | TP (+/-) | TP (+/-) | TP (+/-) | TP (+/-) | TP (+/-) | TP (+/-) | TP (+/-) | |
S1 | 19.68 (+) | U (-) | U (-) | U (-) | U (-) | U (-) | U (-) | U (-) |
S2 | 17.73 (+) | 18.30 (+) | U (-) | U (-) | U (-) | U (-) | U (-) | U (-) |
S3 | 16.73 (+) | U (-) | U (-) | 18.05 (+) | 12.18 (+) | U (-) | U (-) | U (-) |
S4 | 21.66 (+) | U (-) | U (-) | 27.07 (+) | 19.61 (+) | U (-) | 18.29 (+) | U (-) |
S5 | 16.16 (+) | 20.85 (+) | U (-) | 23.80 (+) | 17.13 (+) | U (-) | 15.22 (+) | U (-) |
S6 | 11.20 (+) | 16.79(+) | 17.18(+) | 23.09 (+) | 16.14 (+) | U (-) | 14.28 (+) | U (-) |
S7 | 14.69 (+) | 24.84(+) | 19.70(+) | 24.07(+) | 14.16 (+) | – (+) | 15.23 (+) | 20.12 (+) |
Each Sample+/-, qualitative result; +, positive result; -, negative result. |
Table 6: Table of TP values for detection after infection.
Supplementary Table 1: List of abbreviations. Please click here to download this File.
Supplementary File 1: Respiratory pathogens nucleic acid detection kit instructions. Please click here to download this File.
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'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'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.
The authors have nothing to disclose.
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's Hospital (Grant No. KY012023293).
Bath Incubator(MK2000-2) | ALLSHENG | Provide a constant temperature environment | |
Bronchial lavage fluid collector head | TIANPINGHUACHANG | SEDA 20172081375 | Collecting bronchoalveolar lavage fluid |
Fiberoptic bronchoscope | OLYMPUS | SEDA 20153062703 | 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 |
HR1500-![]() |
Haier | SEDA 20183541642 | Biosafety cabinet |
NAOH | MACKLIN | S817977 | Liquefy viscous lower respiratory tract sample |
Nucleic acid detection kit for respiratory tract pathogens | Capitalbio Technology | SEDA 20173401346 | Testing for bacteria infection |
Nucleic acid extraction reagent | Capitalbio Technology | SEDA 20160034 | For DNA extraction |
RTisochip-W | Capitalbio Technology | SEDA 20193220539 | Loop-mediated Isothermal Amplification |
THERMO ST16R | Thermo Fisher Scientific | SEDA 20180585 | Centrifuge the residual liquid off the wall of the tube. |
Vortex mixer VM-5005 | JOANLAB | For mixing reagent |