Fig 4.5: The detection limit of LAMP (a) and PCR (b) using spiked blood A. baumannii sample. 1 = 5.5×106 CFU/ml; 2 = 5.5×105 CFU/ml; 3 = 5.5×104 CFU/ml. Amplified product is seen for 1-2. 4.2 P. aeruginosa 4.2.1 Optimized LAMP Five temperatures were tested to determine the optimum temperature. These included 60℃, 62℃, 63℃, 64℃and 65℃ (Fig 4.6). Figure 4.6 shows that at 65℃ the reaction had the shortest amplification time. The primer for was P. aeruginosa different from the primers for and This set of primer was A. baumannii K. pneumoniae. combined with loop primers, so the amplification time would be much shorter. Fig 4.7 shows the difference between the amplification with and without loop primer. The whole reaction was run for 60 min at 65℃. With the inclusion of loop primers, the amplification of began at 25 min, loop primer made a 20 min saving of P. aeruginosa the reaction. Compared to the reaction of and this A. baumannii K. pneumoniae, reaction was faster. This shows that the ability of loop primer was to shorten the detection time. Fig 4.6: Comparison of LAMP results for PS19 at different P. aeruginosa temperatures Comparison of LAMP results with or without loop primer on PS19 Fig 4.7:
Fig 4.5: The detection limit of LAMP (a) and PCR (b) using A. baumannii spiked blood sample. 1 = 5.5×106 CFU/ml; 2 = 5.5×105 CFU/ml; 3 = 5.5×104 CFU/ml. Amplified product is seen for 1-2. 4.2 P. aeruginosa 4.2.1 Optimized LAMP Five temperatures were tested to determine the optimum temperature. These included 60℃, 62℃, 63℃, 64℃and 65℃ (Fig 4.6). Figure 4.6 shows that at 65℃ the reaction had the shortest amplification time. The primer for P. aeruginosa was different from the primers for A. baumannii and K. pneumoniae. This set of primer was combined with loop primers, so the amplification time would be much shorter. Fig 4.7 shows the difference between the amplification with and without loop primer. The whole reaction was run for 60 min at 65℃. With the inclusion of loop primers, the amplification of P. aeruginosa began at 25 min, loop primer made a 20 min saving of the reaction. Compared to the reaction of A. baumannii and K. pneumoniae, this reaction was faster. This shows that the ability of loop primer was to shorten the detection time. Fig 4.6: Comparison of LAMP results for P. aeruginosa PS19 at different temperatures Fig 4.7: Comparison of LAMP results with or without loop primer on PS19
DEVELOPMENT_OF_A_RAPID_DETECTION_ASSAY_FOR_POTENTIAL_NOSOCOMIAL_PATHOGENS.docx