Figure 2
Establishment of the quantification method. (A) Distribution of bacteria (CFU/mL) in saline after low-speed centrifugation. The bacterial concentration in the upper half and the lower half were examined after low-speed centrifugation (100×g, 5 min). A total of 2 mL of bacterial suspension (E. coli, S. aureus, K. pneumoniae and P. aeruginosa) in saline was centrifuged, and the colony-forming unit (CFU) value was measured in the upper and lower half (1 mL each). The figure shows the ratio (%) of each half. Error bars indicate triplicate testing. (B) The primer designs. Nested PCR is performed using seven bacterial universal primer sets, and then the seven PCR amplicons (Tm values) are obtained. The pathogenic bacteria are identified using the seven Tm values of region 1 to 7 amplicons, and the bacterial concentration is measured using the region 3 amplicon alone. (C) Standard curves generated using three different primers. We generated standard curves by conducting an analysis of serial dilutions of E. coli DNA using primers as follows: 1st PCR forward primer with no mismatch against E. coli, 1st PCR forward primer with one mismatch against E. coli, and a mix of both 1st PCR forward primers (no mismatch : one mismatch = 1: 1). The 1st PCR reverse primer was the same in all cases. These results indicated that the quantification result using the 1st PCR forward primer with 1 mismatch was around 25% lower than that using the 1st PCR forward primer with no mismatch. In contrast, the quantification result using the mixed 1st PCR forward primers was almost the same as that using the 1st PCR forward primer with no mismatch. Error bars indicate triplicate testing. (D) The comparison of the regression lines generated by serial dilution of a known amount of E. coli DNA using conventional PCR method and nested PCR method. The regression lines calculated for the datum points are shown. The linear correlation between the Ct values and the logarithm of the number of E. coli/PCR tube (R2 value) was > 0.99. Error bars indicate triplicate testing.
