Real-time PCR-based rapid and culture-independent detection of Salmonella in dairy milk - addressing some core issues

Abstract

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. AbstractIn this study, methodologies were developed for cost-effective, rapid and userfriendly 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 AE 0Á5°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 C T value of 37Á17 (SD 0Á43). 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.

Foodborne bacteria in dairy products: Detection by molecular techniques

“…The use of qPCR has provided several advantages over conventional PCR such as quantification, real-time and in situ analyses, in addition to automation (Riyaz-Ul-Hassan et al, 2013). In this technique, the PCR products are detected as they accumulate.…”

“…Milk is a food that has a high chance of contamination by Salmonella spp. (Riyaz-Ul-Hassan et al, 2013), mainly before leaving the farm, usually because of fecal contamination during the milking process (Ahmed and Shimamoto, 2014). Additionally, Salmonella spp.…”

“…These methods have also become valuable tools for investigating foodborne outbreaks and identifying the responsible etiological agents (Riyaz-Ul-Hassan et al, 2013).…”

A multiplex PCR assay for detection of genus Salmonella, subspecies I , and serotype Typhimurium in milk

“…Además de los métodos microbiológicos estándar para la detección de S. enterica en alimentos, se ha diseñado una gran diversidad de ensayos basados en la PCR tanto de punto final como de tiempo real (7,8,9) , algunos de los cuales ya se encuentran disponibles como kits comerciales (7,10) . Aunque los ensayos de PCR en tiempo real para la detección de S. enterica en alimentos tienen gran sensibilidad, rapidez y especificidad (7,8) , los altos costos de implementación y el nivel de capacitación requerido limitan el acceso a dichas técnicas por las instituciones públicas encargadas de la inocuidad de alimentos y vigilancia sanitaria, manteniendo a los ensayos de PCR de punto final como una opción para la detección de S. enterica en alimentos en países en vías de desarrollo como es el caso de México.…”

“…Besides, the standard microbiological methods for detection of S. enterica in foodstuffs, a varied assortment of endpoint and real-time PCR assays have been designed for that purpose (7,8,9) , some of which are available as commercial kits (7,10) . Despite that real-time PCR analyses for detection of S. enterica in foodstuffs are highly sensitive, fast, and specific (7,8) , their elevated costs and the level of training needed for its implementation limits its access by public institutions in charge of food safety and sanitary survey, which makes endpoint PCR assays to be an option for detection of S. enterica in foodstuffs in developing countries as Mexico.…”