F-score = [(fluorescence of the sample – fluorescence of the negative control) / fluorescence of the negative control] x 100

#### Table 5. Sensitivity of the iTPA assays

#### **[Detection of** *Salmonella* **spp. cells in inoculated food samples]**

The detection limits of *Salmonella* spp. inoculated in three food samples are shown in Table 3. In inoculation experiments, the *invA*-based iTPA assay using the serial dilution platforms consistently detected at an initial inoculums level of less than 10 CFU in the pre-enriched food samples (egg yolk, chicken breast, and peanut butter). In Table 4 the results of the Fscore measurement for the iTPA reaction for 60 min at 58°C in the artificially contaminated samples are shown. For statistics, one-way analysis of variance (ANOVA) test was performed (Clarke and Cooke 1998). The mean F-scores for uninoculated peanut butter, egg yolk and chicken breast were 14±4.7, 20±10, and 20±7.2, respectively. The mean F-scores for inoculated peanut butter, egg yolk and chicken breast were 87.34±30.24, 59.09±36.16 and 68.24±26.33(p≤0.001), respectively. The lowest detection limit achieved in this study was a less than 10 CFU per 25 g of food samples.

In this study, we designed a set of DNA-RNA-DNA chimeric primers and a FRET probe to specifically target the *Salmonella* spp. *invA* gene. So, a novel and rapid DNA detection system has been developed which we have termed isothermal target and probe amplification (iTPA). By simultaneously utilizing the dual amplification powers of the target DNA and FRET probe, we have demonstrated that iTPA can be used to rapidly detect less than 10 CFU of *Salmonella* spp. in food samples after pre-enrichment. The four chimeric primers and one FRET probe were designed from five regions of the *Salmonella* spp. *invA* gene coding sequence that are highly specific for *Salmonella* spp. (Table 1). In conclusion, we have developed a DNA detection system which is conveniently performed by requiring only a water bath and a fluorometer and has great potential in applications for hand-held or point-of-care-testing (POCT) diagnostics. The *invA*-based iTPA assay developed in this study is a specific, sensitive, and rapid method for the detection of *Salmonella* spp. in food


samples. This simple method is expected to enable a rapid risk assessment of pathogen contamination of foods at a low cost. (Kim *et al*. 2011)
