Screening Of Specific Targets And Development Of A Real-time PCR Method For The Detection Of Salmonella

In this study 361 candidates for Salmonella-specific targets were successfully identified by comparing the sequences of Salmonella enterica and other prokaryotic genomes using bio-informatic analysis. Primers were designed from 30 randomly selected fragments for end-point PCR detection. The specificity of 15 primer sets was verified by testing against 58 Salmonella strains and 22 non-Salmonella strains, and 6 primer pairs, namely c1 (36.5 fg/PCR, 500 cfu/ mL); c3 (36.5 fg/PCR, 500 cfu/ mL); c20 (36.5 fg/PCR, 500 cfu/ mL); c23 (36.5 fg/PCR, 5×104 cfu/ mL); c24 (36.5 fg/PCR,500 cfu/ mL) and plc (36.5 fg/PCR, 500 cfu/ mL),showed high sensitivity. The PCR method yielded positive results from milk samples that were artificially contaminated with Salmonella Typhimurium (as low as 8 cfu/25mL milk) within 6-8 hours of non-selective enrichment. The enrichment time was shortened to 2-4 hours when the samples were heavily contaminated ( >104 cfu/25mL milk).One of the verified Salmonella specific fragments was targeted to generate a minor groove binding TaqMan probe and the corresponding primer set for real-time PCR detection. An internal amplification control obtained by DNA random shuffling technology was included in the real-time PCR assay to indicate false negative results caused by PCR inhibitors. The real-time PCR method, incorporated with the IAC, demonstrated a high specificity when tested against 40 Salmonella strains and 24 non-Salmonella strains. Detection limits were 18.6 fg genomic DNA /PCR for Salmonella Enteritidis (CDC H3526) and 40.2 fg/PCR for Salmonella Typhimurium (CDC G7601). In the presense of a natural background flora enriched from chicken breast samples, the detection limit was as low as 130 cfu/mL, and the quantitation results of the real-time PCR assay were in accordance with the actual amount of the spiked Salmonella cells prior to DNA extraction. The real-time PCR method was able to detect as low as 5 cfu/10g chicken sample or 1 cfu/ 10g liquid egg sample after a 6-hour non-selective enrichment. Twenty-six out of 27 peanut butter samples, artificially contaminated with as low as 3 cfu/10g sample, were confirmed positive by this real-time PCR sysytem, exhibiting a 97.2% consistency with the standard culture methods.