In this study, methodologies were developed for cost-effective, rapid and user-friendly culture-independent detection of Salmonella in milk by real-time PCR. The SYBR Green-based real-time PCR assay was standardized with primers targeting the Salmonella enterotoxin gene (stn) that have been earlier used for its detection by conventional PCR. Inclusivity tests generated the specific amplifications with a Tm corresponding to 81 ± 0·5°C. The specificity of the reaction was evaluated with a panel of 36 non-Salmonella strains. Standard curves generated, with different number of cells of this organism in milk, depicted the detection of five cells with a CT value of 37·17 (SD 0·43). To make the assays user-friendly and suitable for field applications, protocols were also established for the immobilization of the SYBR Green reaction mixes in the reaction tubes. The immobilized master mixes were stable at 25°C for 4 months and at 8°C for over 6 months. Total DNA was prepared from 150 samples of full-fat dairy milk and subjected to real-time PCR detection wherein 31 samples tested positive for Salmonella. The time of analysis was <5 h.
Significance and Impact of the Study
With the familiarity of the polymerase chain reaction (PCR) in diagnostic microbiology, it is becoming increasingly possible to use the nucleic acid amplification methodologies for rapid detection of pathogens under field conditions. However, it is imperative to develop assays that are culture-independent, cost-effective and user-friendly. The methodologies developed in this study, particularly the immobilization of the reaction components on the vessels and total DNA extraction from milk, may help to facilitate the use of this technology under field settings. The protocols may thus facilitate the routine culture-independent detection of pathogens, in particular Salmonella, in dairy milk.