By: Mutsyahidan AMA1, Rahayu WP2,3, Nurjanah S2,3
1Department of Agricultural Product Technology, Polytechnic of Gorontalo, Indonesia
2Department of Food Science and Technology, Bogor Agricultural University
3SEAFAST Center, Bogor Agricultural University
Listeria monocytogenes is an important foodborne pathogen that can cause serious human listeriosis. The aim of this study was to optimize real-time PCR method for L. monocytogenes detection in indigenous Indonesian snack (pempek). DNA isolation of L.monocytogenes was conducted by comparing three extraction methods: phenol:chloroform, heating, and commercial kit (QIAamp DNA Blood Mini Kit). DNA isolate was amplified by PCR using two variables, which were LIM 2 and LIMRE primers for iap gene detection, and DG69 and DG74 primers for hlyA gene detection. Phenol:chloroform method showed the best extraction result, while DG69 and DG74 primers showed more specific result on PCR compared with LIM 2 and LIMRE primers. The running conditions gave specific amplification curve until 27th cycle. DNA detection in L. monocytogenes culture generated standard curve equation of Ct = 37.9 – 3.11 C with limit of detection (LOD) at 3.2 x 103 CFU/mL, while DNA detection in pempek generated standard curve equation of Ct = 41.03 – 3.69 C with limit of detection (LOD) at 6.3 x 103 CFU/g. Real-tie PCR with DG69 and DG74 primers could be considered as a reliable method for specific and sensitive detection of L. monocytogenes.
Keywords : Detection, Indigenous snack, Listeria monocytogenes, Real-time PCR.
Published at Malaysian Journal of Microbiology. 12(2): 177-181. 2016. ISSN (print) 1823-8262, ISSN (online): 2231-7538