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Swabs and sponges were pre-moistened with Dey-Engley (D/E) Neutralizing broth and
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used to sample the entire sampling area in both a horizontal and vertical motion. Swabs
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and sponges were held at room temperature for 2 h prior to enrichment in order to
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simulate sample transfer time from a collection site to a laboratory.
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VIDAS LPT method (surfaces)
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Pre-enrichment: Ten mL of LPT broth (at room temperature) was added to each swab or
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100 mL LPT broth (at room temperature) was added to each sponge. Samples were
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homogenized for 2 minutes then incubated at 30 ± 1°C for 22-30 h. After incubation,
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0.5ml of LPT broth was transferred to the sample well of the LPT strip. The strip was
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heated for 5 ± 1 min on Heat and Go then cooled 10 min. prior to performing the VIDAS
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LPT assay according to manufacturer’s instructions.
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Confirmation: After 20-24h 0.1ml of the LPT enrichment was transferred to 10ml Fraser
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both (FB) and incubated at 35±1°C for 48 h. Any FB showing blackening (24-48h) was
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streaked to MOX agar. In addition, approximately 0.1ml of the LPT broth (22-30 h) was
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streaked directly to Modified Oxford agar (MOX) and ALOA agar, then incubated for 24-
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48h at 35±1°C. At least one typical colony per sample/media was streaked to Horse blood
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overlay agar (HLA) and confirmed using standard biochemical tests.
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USDA FSIS Reference Method (surfaces)
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225 mL UVM broth was added to each sponge and 10 mL UVM broth was added to each
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swab. After homogenization, UVM enrichments were incubated for 20-24h at 30
±
2
°
C.
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After 20-24h 0.1ml of the UVM enrichment was transferred to 10ml Fraser both (FB) and
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incubated at 35±1°C for 48 h. Any FB showing blackening (24-48h) was streaked to
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MOX agar. In addition, approximately 0.1ml of the UVM broth (20-24h) streaked
24
directly to Modified Oxford agar (MOX) and ALOA agar, then incubated for 24-48h at
25
35±1°C. At least one typical colony per sample/media was streaked to Horse blood
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overlay agar (HLA) and confirmed using standard biochemical tests (FDA BAM).
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Data Analysis
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The data were analyzed according to AOAC guidelines (6). Comparison of data for the
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LPT method and the reference method using Chi-square test at the 5% level was
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conducted for unpaired data (Mantel-Haenszel test). The Chi-square analysis must show
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that there was no significant difference between the two methods at the 5% level or that
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the LPT method was significantly better than the reference method.
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Additionally, data were analyzed using Probability of Detection (POD) statistics.
POD is
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the proportion of positive analytical outcomes for a qualitative method for a given matrix
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at a given analyte level or concentration. POD is concentration dependent. Several
POD
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measures were calculated; POD
R
(reference method POD), POD
C
(confirmed candidate
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method POD), POD
CP
(candidate method presumptive result POD) and POD
CC
(candidate
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method confirmation result POD).
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