-specific primers. Under this optimized m-PCR condition, three types of PCR were performed: uniplex (Fig. 1, lanes 1–3), duplex (Fig. 1, lanes 4–6), and triplex (Fig. 1, lane 7). Each PCR result exhibited high specificity and sensitivity of target products and the amplicon size was the same as the expected value. Each

target genomic DNA was prepared from 1 mL of pure culture bacteria containing 7.33 × 107 copies, and was diluted 10-fold until 7.33 × 100 copies. In a uniplex PCR, the Campylobacter spp.-specific primer pair was more sensitive than the other two primer pairs in detecting target microorganisms. The detection limit of C. jejuni was 7.33 × 101 copies, while there were 7.33 × 102 copies of E. coli O157:H7 and S. Typhimurium in pure culture samples (Table 3). In contrast to uniplex PCR, m-PCR showed detection limits of 7.33 × 103 copies in mixed

culture sample detection of the learn more three bacteria due to primer competition as well as dimer formation (Fig. 2a) and all results were based on triplicate experiments. Watershed samples were collected from a local farm and analyzed using traditional selective media to confirm whether samples were contaminated naturally. Samples were aliquoted CHIR-99021 datasheet and analyzed immediately using the conventional plate method and PCR and also analyzed after 7 days of storage at 4 °C. By conventional plating, the number of C. jejuni, E. coli O157:H7, and S. Typhimurium in samples stored for 7 days decreased by 1–2 logs compared with the initial inoculation levels (Table 4). Campylobacter jejuni was reduced from 5.3 ×

109 to 2.2 × 107 CFU mL−1, E. coli O157:H7 was reduced from 9.3 × 108 to 6.7 × 107 CFU mL−1, and S. Typhimurium was reduced from 3.2 × 109 to 4.3 × 108 CFU mL−1 (Table 4) To evaluate the m-PCR assay, different concentrations of each bacteria were inoculated into the watershed samples; 0-day samples of C. jejuni contained 5.3 × 109–5.3 × 102 CFU mL−1, E. coli O157:H7 contained 9.3 × 108–9.3 × 101 CFU mL−1, S. Typhimurium Dimethyl sulfoxide contained 3.2 × 109–3.2 × 102 CFU mL−1 and 7-day samples [C. jejuni (2.2 × 107–2.2 × 100 CFU mL−1), E. coli O157:H7 (6.7 × 107–6.7 × 100 CFU mL−1), S. Typhimurium (4.3 × 108–4.3 × 101 CFU mL−1)]. Uniplex and multiplex PCR results showed that there was no obvious difference between 0- and 7-day samples (Fig. 2b and c) in detection limitation. Only the detection limitation of C. jejuni was decreased by fourfold in a uniplex PCR (data not shown). Purified genomic DNA of C. jejuni, E. coli O157:H7, and S. Typhimurium were used to design standard curves and the calculated DNA copy numbers ranged from 7.33 × 107 to 7.33 × 101 copies μL−1. Only the C. jejuni standard curve could be constructed to start at 7.33 × 100 copies μL−1 due to the high sensitivity of the primer pair. As a result, the lowest copy number was determined as the detection limit in pure culture DNA for each bacterium. The melting temperature of C.