**4.2 Results and discussion**

#### **[Inclusivity and exclusivity of the iTPA assay]**

The *Salmonella* spp. *invA*-based iTPA assay, which required only a water bath and the RF-1000 fluorescent reader successfully detected 10 *Salmonella* spp. strains while showing negative results for 40 non-*Salmonella* spp. strains (Table 1), indicating that the *invA-*based iTPA assay was specific for *Salmonella* spp.. The PCR assay using iTPA outer primers yielded amplicons of the expected size (117 bp) for all 10 Salmonella spp. strains. (data not shown) Two sample t-tests were performed for negatives and positives in pure culture. The mean F-score of the negatives was 3.97±0.44 and the mean F-score of the positives was 82.9±6.1 (p≤0.001, data not shown). For a rigorous exclusivity comparison, the positive strains were used at a low concentration of the genomic DNA (1pg, ca. 102 CFU) as the template while the negative strains were used at very high concentration of the genomic DNA (ca. 105 CFU). Neither false positive nor false negative results for the 50 bacterial

Studies on PCR-Based Rapid Detection Systems for Salmonella spp. 435

samples. This simple method is expected to enable a rapid risk assessment of pathogen

1.7±0.4 x 103 1.7±0.4 x 102 1.7±0.4 x 101 1.7±0.4 x 100 1.7±0.4 x 10-1 un-inoculated 1.7±0.4 x 103 1.7±0.4 x 102 1.7±0.4 x 101 1.7±0.4 x 100 1.7±0.4 x 10-1 un-inoculated 1.7±0.4 x 103 1.7±0.4 x 102 1.7±0.4 x 101 1.7±0.4 x 100 1.7±0.4 x 10-1 un-inoculated 87±5.7 86±6.6 82±12 82±13 39±40 14±4.7 72±4.0 73±4.0 69±7.3 44±3.4 11±26 20±10 72±3.6 72±2.3 71±4.8 56±19 34±29 20±7.2

Food samples Dilution No. of Bacteria#(CFU) F-score

10-5 10-6 10-7 10-8 10-9

10-5 10-6 10-7 10-8 10-9

10-5 10-6 10-7 10-8 10-9

Table 6. Detection limits for inoculated food samples by iTPA assay

monocytogenes. Appl. Environ. Microbiol 61, 3724-3728.

hybridization probes. J. Clin. Microbiol 39, 370-374.

Multiplex PCR. Appl. Envir. Microbiol 65: 4688-4692.

chicken feces. Amer. micro 71: 4214-4219.
