In this study, loop-mediated isothermal amplification (LAMP) assay in conjunction with calcein for visualized detection of Mycobacterium tuberculosis (MTB) was established. Firstly, four LAMP primers were designed according to the region of 16S rDNA sequences of MTB. Secondly, clinical sputum samples were collected, decontaminated and their DNA was extracted. Thirdly, standard MTB strains were used to evaluate the specificity and sensitivity of LAMP. At the same time, electrophoresis was used for products detection and calcein was used for visualized verification. At last, Chi-squared test function in SPSS 17.0 software was used for consistency evaluation of LAMP assay as compared with the gold standard (culture method). Results showed that there was no nonspecific amplification appeared in the specificity assay and the detection limit was 10 copies/tube in the sensitivity assay. In addition, visualized method by calcein had a comparable sensitivity with that of electrophoresis method. After evaluation of clinical practicability, the sensitivity of LAMP was calculated as 94.74% and the specificity was 90%, respectively. And Chi-squared test showed that LAMP and culture method had no statistic difference, and the two methods were in good consistency (P>0.05). In conclusion, LAMP assay introduced in our study has the characteristics of high efficiency and visualized detection so that this technique has great application prospects in the resource-limited environment, such as work field and primary care hospitals.
Objective To evaluate the clinical value of loop-mediated isothermal amplification chip (LAMP) in pathogen detection for acute exacerbation of chronic obstructive pulmonary disease (AECOPD). Methods We retrospectively analyzed qualified lower respiratory tract specimens from AECOPD patients treated at West China Hospital of Sichuan University between February 2021 and December 2022. Both LAMP and conventional culture methods were performed simultaneously to compare their positive detection rates, pathogen profiles, and diagnostic consistency (Kappa test). Subgroup analysis was further performed based on the site of infection [community acquired pneumonia (CAP) vs. hospital acquired pneumonia (HAP)]. Results A total of 159 patients were included. Among the 159 specimens, the overall positive rate of LAMP was significantly higher than that of the culture method [36.5% (58/159) vs. 30.2% (48/159)]. Significant differences in positive rates were observed for Acinetobacter baumannii (13.8% vs. 11.3%) and Pseudomonas aeruginosa (9.4% vs. 7.5%). The LAMP chip method detected mixed infections in 13 cases and additionally identified 6 cases of Mycobacterium tuberculosis complex. Consistency analysis between the two methods for respiratory pathogen detection showed statistically significant agreement for Escherichia coli, Staphylococcus aureus, methicillin-resistant Staphylococcus aureus (MRSA), Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, Stenotrophomonas maltophilia, and Haemophilus influenzae (P<0.05). In the CAP subgroup, only the LAMP method exclusively detected Mycobacterium tuberculosis complex (5.6%), Streptococcus pneumoniae (2.8%), Staphylococcus aureus (0.9%), and Escherichia coli (1.9%). In the HAP subgroup, only the LAMP method exclusively detected MRSA (3.8%). Significant differences in positive rates between the two methods for multiple pathogens were observed in both CAP and HAP groups (P<0.05). Conclusions For pathogen detection in AECOPD patients, the LAMP method offers rapid (only 2 hours), simple and efficient testing, particularly valuable for mixed infection screening. It shows advantages in detecting fastidious pathogens in community-acquired pneumonia, and providing guiding significance for early precise anti-infective therapy.