Development of an enhanced LAMP-based method for early identification of Streptococcus pneumoniae through LytA gene amplification.

INTRODUCTION

Streptococcus pneumoniae is a leading cause of invasive bacterial disease in developed and developing countries. The LytA gene acts as a key pneumococcal virulence factor. No simple, cost-effective technology exists to detect this gene. In this study, we developed and validated a quick and visible LAMP assay targeting the LytA gene for pneumococci detection.

METHODOLOGY: Visual LAMP assay was performed to amplify the S. pneumoniae LytA gene using five primers. S. pneumoniae ATCC® 49619™ was employed as a positive control, and 19 pathogenic bacterial strains without the LytA gene and 20 clinical isolates with the gene were utilized to test LAMP assay specificity. To assess sensitivity, the visual LAMP assay was compared to PCR using ten-fold serial dilutions of Pneumococcal ATCC DNA. The clinical sensitivity and specificity of the enhanced LAMP assay were tested with 25 pneumonia-suspected clinical samples. In addition, standard microbial culture and PCR analysis were also performed for comparison purposes.

RESULTS

After 60 min of incubation at 65 °C, the S. pneumoniae ATCC strain and all 20 clinical pneumococci with defined primers amplified LytA target DNA and gave a color change from pink to yellow. DNA amplification was absent in 19 pathogenic bacterial strains without LytA genes under the same circumstances. The detection limit for the improved LAMP assay was 6 CFU/mL (0.425 fg/μL). Given conventional microbiological culture as the gold standard, the PCR assay provided two false positives, whereas the LAMP assay gave no false positives with 25 clinical samples. The LAMP assay exhibited 100 % sensitivity, specificity, PPV and NPV (95 % CI) utilizing the conventional culture method as a gold standard.

CONCLUSION

The enhanced LAMP assay visually detects the pneumococcal LytA gene faster, simpler, more sensitively, and more specifically than PCR and microbiological methods. This enhanced LAMP method is suited for low-resource population screening and diagnosis.