[4-1015-C] Food Safety (1)

[4-1015-C-04] Sensitivity Comparison of Standard and real-time PCR Assay for Detection Salmonella Typhimurium and Enteritidis in Indonesian Chicken Carcasses

*siti nurjanah^1,2, Winiati Puji Rahayu^1,2, Ratih Dewanti-Hariyadi^1,2 (1. Department of Food Science & Technology, Bogor Agricultural University (IPB University)(Indonesia), 2. SEAFAST Center, Bogor Agricultural University (IPB University)(Indonesia))

Keywords:chicken carcasses, real-time PCR, Salmonella Enteritidis, Salmonella Typhimurium, sensitivity

Salmonella is a pathogenic bacterium that can cause serious illness to humans. Chicken carcasses have been reported contaminated by Salmonella, especially S. Typhimurium and S. Enteritidis. Rapid and sensitive method for detection and differentiation of both species is required. The objective of this study was to compare the sensitivity of standard and real-time PCR (rt-PCR) assay for detection and differentiation two Salmonella serovars using pre-enrichment and enrichment treatment. Primer from invA gene, specific Typhimurium protein (STM) and specific Enteritidis virulence plasmid (Prot6E) sequences were used for marker of Salmonella genus, S. Typhimurium and S. Enteritidis respectively. Sensitivity determination was carried out by artificially contaminated of each Salmonella serovars into chicken carcasses approximately amount of 10⁵ CFU/mL. After contaminated, DNA was extracted by chelex100 method and subsequently one-tenth dilution up to lowest concentration. Sensitivity was determined based on the lowest amount of DNA remain detected then converted to the cells number. The result showed that rt-PCR was more sensitive for genus detection. Without pre-enrichment step, the limit detection of rt-PCR for Salmonella genus, S. Typhimurium and S. Enteritidis were equivalent with 3.8 x 10¹ cells, 4.1 x 10³ cells, and 2.6 x 10⁴ cells respectively, meanwhile the standard PCR for each were equivalent with 5.3 x 10⁴ cells, 4.0 x 10⁴ cells, 4.1 x 10³ cells respectively. Both of methods were applied to detect 20 Salmonella-contaminated chicken carcasses; rt-PCR assay can detected all of positive samples, whereas standard PCR showed 3 false negatives results. However, both of method showed similar results for detection serovars S. Typhimurium and S. Enteritidis. Pre-enrichment step in buffer phosphate for 8 hours increased the sensitivity of standard PCR until less than 10 cells even for two serovars detection. It’s concluded that analysis by standard PCR required pre-enrichment step for obtaining the sensitive result.