Total Collaborative Study Protocol_Solus One Salmonella v1 1

Robustness Study 

This study was carried out with the inoculation of a food matrix, using a three‐parameter factorial 

design, where both high and low parameter values were incorporated, plus a combination of the 

nominal parameter values. All other parameters remained unchanged. 

Parameters to be varied included; sample incubation time (18, 26 h), conjugate volume (90, 110 µL), 

and TMB substrate incubation time (20, 40 min). Recommended conditions analyzed were 20–22 h 

sample incubation, 100 µL conjugate volume and 30 min TMB substrate incubation. 

For the robustness study evaluation of Solus One  Salmonella , a  Salmonella Typhimurium NCTC 5711 

strain was cultured in non‐selective BHI broth for 20 ± 2 hours at 37 ± 1°C. The starter culture was 

diluted to approximately 10 1  CFU/mL in sterile BHI broth and used to inoculate ten 375 g test portions of 

raw beef (trim, >30% fat content) matrix enriched with 1,125 mL supplemented BPW medium pre‐

warmed to 37°C. In addition, ten–375 g test portions of non‐inoculated raw beef (trim, >30% fat 

content) matrix were also enriched with 1,125 mL supplemented BPW medium pre‐warmed to 37°C. 

The selected raw beef trim matrix was not naturally contaminated with target analyte and aerobic plate 

count was 2.3 x 10 5  CFU/g. The inoculated and non‐inoculated test samples were subsequently 

incubated for 26 hours at 41.5 ± 1°C. Several 1 mL aliquots were removed from each test sample at 18, 

20–22 (representative of the nominal condition) and 26 h.  

The 18, 20–22 and 26 h inoculated test samples were diluted in sterile supplemented BPW 

enrichment media to the LOD 50

of the method to obtain fractional detection levels. The 18, 20–22 and 

26 h non‐inoculated test samples were used without dilution. 

Where applicable for inoculated and non‐inoculated samples, ten 18 h undiluted and diluted 

aliquots were used for factorial combinations 1 and 2, with another ten 18 h undiluted and diluted

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