**[iTPA specificity and detection limits]**

Fifty bacterial strains were used to determine the iTPA specificity. DNA templates were prepared from bacterial cultures and aliquots were subjected to the iTPA assay. For *Salmonella* strains, the genomic DNA was isolated from the overnight culture grown in LB

recognized five distinct regions of the target sequence were designed using the DNASTAR software (Maison, WI). Oligonucleotide sequences and locations of the primers and the probe are shown in Table 1. The DNA primers were synthesized by Genotech (Daejeon, South Korea). The chimeric primers and a FRET probe were synthesized by IDT (San Diego, CA). The iTPA reaction mix in a 20 *μl* volume consisted of the following: 10 mmol / L of Tris-HCl (pH 8.5), 22 mmol / L of MgSO4, 10 mmol / L of KCl, 10 mmol / L of (NH4)2SO4, 0.05 mg ml / L of acetylated BSA, 3 mmol / L of DTT, 0.4 mmol / L of deoxynucleotide triphosphate (dNTP), 0.22 mol / L of each outer primers, 2.2 mol / L of each inner primers, 100 nmol / L of the FRET probe, 5 units of *bst* polymerase (NEB, Ipswich, MA), 5 units of RNase H (Epicentre, Madison, WI), 6 units of RNase inhibitor (Solgent, South Korea), and 2 l l of DNA template (2 l of sterilized water was used for a negative control). The iTPA reaction mix was incubated at 58°C for 60 min in a water-bath and then cooled to room temperature. After a quick spin-down, the reaction tube was inserted into a RF-1000 fluorescent reader (Raplegene, Inc., Sungnam, South Korea) to read the relative RFU (fluorescence relative unit) signal. The RF-1000 fluorescent reader calculates the F-score and it is display on the LCD window. The result was interpreted as a *Salmonella* spp. positive if the F-score was ≥35 or a *Salmonella* spp. negative if the F-score was < 35. This cut-off value was determined using uninoculated food samples that had also undergone cultural pre-enrichment. F-scores of uninoculated egg yolk samples and chicken meat samples were 20±9.5 and 20±7.2 respectively

(p≤0.001). The equation used to calculate the F-score is the following:

may be contaminated, we used sterile water as the negative control.

CCT GAT CGC ACT GAA TAT C CGA AAG AGC GTG GTA ATT AAC

TAT TGG TGT TTA TG

AAA CGT TGA A

DABCYL

**[iTPA specificity and detection limits]** 

Outer forward Outer reverse Inner forward

Inner reverse FRET probe

F-score = [(fluorescence of the sample – fluorescence of the negative control) / fluorescence of the negative control] x 100. Sterilized water was used for the negative control reaction instead of the extracted nucleic acid from inoculated food samples. Since uninoculated food samples

Name Sequence (5'-3') Position#

CGA TGA CTG ACT ATA CAA GrUrA rCrGC TGG CGA

99-117 195-215 121-131

174-191 146-170

CTA GTA CAT GAA GCT rArArA rGAC CGC AGG

FAM-CGT TCT ACA TTrG rArCrA rGrAA TCC TCA G-

Fifty bacterial strains were used to determine the iTPA specificity. DNA templates were prepared from bacterial cultures and aliquots were subjected to the iTPA assay. For *Salmonella* strains, the genomic DNA was isolated from the overnight culture grown in LB

Table 4. iTPA primers and FRET probe used in this study to detect Salmonella spp.

medium and then quantified. One picogram of genomic DNA was used as the template. For non-*Salmonella* strains, the genomic DNA was isolated from the overnight culture grown in LB medium and then 2 L of the DNA extraction TE buffer solution was used. Specificity tests were repeated 10 times. To determine iTPA detection limits, serial 10-fold dilutions of a mid-log phase *S. typhimurium* KCTC2515 culture (*ca.* 108 CFU) grown in LB broth were prepared in PBS and quantified using the standard plating method. The detection limit tests were repeated 10 times and the lower limits of detection (CFU per assay) were reported.
