Isothermal and Sensitive Identification of <i>Streptococcus pneumoniae</i> Using Loop Mediated Isothermal Amplification Assay

Abstract

Background: As a major pathogen, Streptococcus pneumoniae is responsible for fatal diseases and is deemed to be a major public health problem in the developing countries. Due to its high mortality rate, rapid detection of pneumococci is necessary in medical centers with limited equipment. Loop-mediated isothermal amplification (LAMP) assay is a rapid method to detect bacterial pathogens. Herein, the current study developed a LAMP assay based on the choline-binding protein M (cbpM) gene to detect pneumococci isolates. Methods: A set of 4 primers was designed using the sequence of cbpM, and a LAMP assay was conducted using a different ratio of inner primers to outer primers, dNTPs, and MgSO4 concentrations. The sensitivity of assays was calculated according to 30 clinical isolates of S. pneumoniae. The specificity of primers was also evaluated using 7 non-pneumococcal species. The detection limits of the LAMP assay were also compared with those of the polymerase chain reaction (PCR) assay using a 10-fold serial dilution of the DNA. Results: Optimal temperature and time for the LAMP assay were 62°C and 1 hour, respectively. Its detection limit was only 5 copies of DNA, compared to 50 copies for PCR. When LAMP was tested on 7 non-pneumococcal species, no amplification was observed. Similar to PCR, all 30 S. pneumoniae isolates were detected using the LAMP assay, which showed 100% sensitivity. Conclusions: The LAMP assay is a favorable tool for the rapid detection of pneumococci, and can be employed in medical centers with limited equipment.