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Non-stochastic sampling error in quantal analyses for Campylobacter species on poultry products

Peter Irwin, Sue Reed, Jeffrey Brewster, Ly Nguyen, Yiping He
Analytical and Bioanalytical Chemistry 2013 v.405 no.7 pp. 2353-2369
Campylobacter coli, Campylobacter jejuni, DNA primers, bacteria, bacterial contamination, breasts, chickens, detection limit, fluorescent dyes, food contamination, food pathogens, polymerase chain reaction, poultry products, quantitative analysis, sampling, stochastic processes, thighs, variance, wings
Using primers and fluorescent probes specific for the most common foodborne Campylobacter species (C. jejuni = Cj and C. coli = Cc), we developed a multiplex, most probable number (MPN) assay using quantitative PCR (qPCR) as the determinant for binomial detection: number of p positives out of n = 6 observations each of 4 mL (V) per dilution. Working with thrice frozen-thawed (to minimize native bacteria) chicken washes spiked with known levels of both Cj and Cc, we found that about 20% of the experiments had a significant amount of error in the form of either greater than 25% MPN calculation error ('e) and/or a low apparent recovery rate (R less than 1 = MPN observed ÷ CFU spiked). Assuming such errors were exacerbated by an excessively small n, we examined computer-generated MPN enumeration data from the standpoint of stochastic sampling error (') and found that such binomial-based assays behave identically to Poisson-based methods (e.g., counting data) except that fewer technical replicates (n) appear to be required for the same number of cells per test volume (u). This result implies that the qPCR detection-based MPN protocol discussed herein should accurately enumerate atest population with a u = 1 using n = 6 observations per dilution. For our protocol this equates to = 8 cells per 400-500g of sampled product. Based on this analysis, the error rate we saw in spiked experiments (where u >> 1) implied a non-stochastic source. In other experiments we present evidence that this source was, at least in part, related to the cell concentration step. We also demonstrate that such errors were lessened (from ~ 20% to ~10%) at lower Campylobacter levels (u = 40) as this would more likely exist in nature. Using this protocol we were able to quantify 14 to 1226 MPN per 450g of naturally contaminated chicken for skinless chicken pieces and 18 to 244 MPN per 450g for chicken wings, breasts, legs, and thighs (skin on) whereupon about 50% of the 29 samples tested negative. Four of these chicken wash samples did have substantially lower Campylobacter levels (1.24 to 6.11 MPN per 450g) which might be better enumerated using n = 12 or higher. However, we demonstrated that the limit of this protocol diminishes greatly for n > 6 because one is ever more diluting the sample, or lessening V, to achieve the requisite n.