The bacterial species Daporinad cell line used in this study were S. aureus, S. pneumoniae, S. suis, S. agalactiae, N. meningitidis, H. influenzae and E. coli. The nucleotide sequences of the 16S rRNA genes of these bacteria were retrieved and aligned to design broad range specific LAMP assay primers using explorer version 4 (Eiken Chemical Co., Ltd). We could not design any broad range specific LAMP assay primers for all the seven bacteria due to high level of variation in the target 16S rRNA gene among species (Fig. 1a). Next, we repeatedly aligned the target gene and designed broad range specific LAMP assay
primers each time removing each species. However, no broad range specific LAMP assay primers were found for the detection of any set of more than four bacterial species. Finally, we successfully designed a set of broad range specific LAMP assay primers for the detection of four species including S. aureus, S. pneumoniae, S. suis and S. agalactiae (Fig. 1b). The name, positions Dabrafenib mw and nucleotide sequences of all four primers are shown in Fig. 1b and Table 1. The DNA sequence alignment of 16S rRNA gene of these four species indicated a low variation among these
species. The sensitivities of the broad range LAMP assay were performed by running 10-fold serial dilutions of target bacteria (from 107 to 100 CFU mL−1). The detection limit was 100 CFU mL−1 of S. pneumoniae by both real-time turbidimeter and electrophoresis of LAMP products, and 10 000 CFU mL−1 by conventional PCR method (Fig. 2). Similarly, the broad range LAMP assay detected S. suis, S. agalactiae Progesterone and S. aureus at 100 CFU mL−1, while conventional
PCR assay only detected these bacteria with more than 104 CFU mL−1 (Table 2). The results of all the positive samples detected by the LAMP assay were achieved within 60 min. The specificity of the LAMP assay was evaluated by cross-reactivity test using DNA extracted from N. meningitidis, H. influenzae and E. coli. There were ladder-like products amplified from S. pneumoniae, S. suis, S. agalactiae and S. aureus but not from N. meningitidis, H. influenzae and E. coli cultures (Fig. 3), suggesting that the broad LAMP assay was specific for S. pneumoniae, S. suis, S. agalactiae and S. aureus. LAMP products were further explored by visual inspection based on the intercalation of fluorescent dye SYBR Green I into amplified DNA. As shown in Fig. 4, the product of positive reaction became visible under ultraviolet lamp and was green colour under naked eye, while the negative product was not seen under ultraviolet lamp and remained orange colour under day light. To identify bacterial species, the LAMP product was digested with specific restriction enzyme and analysed by gel electrophoresis. After digested with DdeI, all LAMP products were digested into several fragments. Staphylococcus aureus gave five bands at 55, 150, 197, 230 and 263 bp (Fig.