Development of Polymerase Chain Reaction assay protocol for assessment Salmonella sp. in cow raw milk

Abstract

Salmonella sp. is a pathogenic bacterium that may associated with acute diarrhoea in human. These bacteria may be transmitted in a variety of ways, including consumption of contaminated cow raw milk. Salmonella sp. is troublesome to assessment due to methodological restrictions. The aim of this study was to development the protocol of Polymerase Chain reaction (PCR) assay for Assessment Salmonella sp. in cow raw milk. This method is comprised of (1) biochemical assay and (2) bacterial DNA purification from Selenite Cystine Broth and XLD Xylose Lysine Deoxycholate culture using Wizard® Genomic DNA Purification Kit followed by PCR detection. The biochemical assay is divided into several stages, namely bacterial isolation, Gram staining and conventional biochemical tests. The PCR optimization was done with Salmonella sp. The oligonucleotide primer invasion protein (invA) gene (F: 5’-TCGTCATTCCATTACCTACC-3’; R: 5’-AAACGTTGAAAAACTGAGGA-3’) were used for targeting the diagnosis of Salmonella at the genus level. In biochemical tests, Salmonella sp. results showed posit if result of catalase, oxidase, citrate, TSIA, lactose-sucrose-mannitol fermentation, urea, and methyl-red. Conversely, negative result for Voges-Proskauer. PCR protocol consist of 30 PCR cycles with initial denaturation at 94°C for 45 seconds, denaturation at 94°C for 20 seconds, annealing at 57°C for 15 seconds, extension at 72°C for 15 seconds and finally at 72°C for 2 minutes. The conventional method detection results obtained as many as 9 positive samples Salmonella sp. and the PCR method obtained 7 positive samples Salmonella sp. In conclusion, our results indicate that the developed protocol would be utilized as a routine analysis for monitoring cow raw milk contamination and the protocol of the PCR technique provides results at a handful of time required by the biochemical assay (24 hours compared to 2–3 days).