Standardization of a rapid quadruplex PCR method for the simultaneous detection of bovine, buffalo, Salmonella spp., and Listeria monocytogenes DNA in milk

J.S Lima,C.M Moraes,P.F.M Sousa,M.C.S Dufossé,J.B Silva,G.V.F Cardoso,A.M.B.P Rosa,T.B Roos,A.P.P.O Sampaio

doi:10.1590/1678-4162-12218

Abstract

ABSTRACT The objective of the present study was to Standardize a Polymerase Chain Reaction (PCR) protocol for the authentication of bovine and buffalo milk, and to detect the presence of Salmonella spp. and Listeria monocytogenes. For this, the target DNA was extracted, mixed, and subjected to a PCR assay. Milk samples were defrauded and experimentally contaminated with microorganisms to assess the detection of target DNA at different times of cultivation, bacterial titers, and concentration of genetic material. In addition, the protocol was tested with DNA extracted directly from food, without a pre-enrichment step. The proposed quadruplex PCR showed good accuracy in identifying target DNA sequences. It was possible to simultaneously identify all DNA sequences at the time of inoculation (0h), when the samples were contaminated with 2 CFU/250mL and with 6h of culture when the initial inoculum was 1 CFU/250mL. It was also possible to directly detect DNA sequences from the food when it was inoculated with 3 CFU/mL bacteria. Thus, the proposed methodology showed satisfactory performance, optimization of the analysis time, and a potential for the detection of microorganisms at low titers, which can be used for the detection of fraud and contamination.

Highlights

Concerns regarding the quality and safety of food have increased due to scenarios in which several products, especially milk, are targets of contamination by pathogenic microorganisms and adulterations (Souza et al, 2011; Mullan, 2019; Farah et al, 2021)
Figure 1. 1.5% agarose gel showing the presence of DNA fragments of bovine (346 bp), buffalo (220 bp), Salmonella spp. (429 bp) and Listeria monocytogenes (702 bp) origin, obtained from quadruplex Polymerase Chain Reaction (PCR) amplification
M: 100 bp molecular size marker; Bb: reaction only with buffalo DNA (Bubalus bubalis); Bt: reaction with bovine DNA (Bos taurus); S: reaction with Salmonella sp.; Lm: reaction with Listeria monocytogenes DNA; Pool: reaction with the mixture of DNAs obtained from each species; Ch: reaction with Caprahircus DNA; Ec: reaction with Escherichia coli DNA; Sa: reaction with Staphylococcus aureus DNA; C-: negative reaction control

Summary

Introduction

Concerns regarding the quality and safety of food have increased due to scenarios in which several products, especially milk, are targets of contamination by pathogenic microorganisms and adulterations (Souza et al, 2011; Mullan, 2019; Farah et al, 2021). Milk and dairy products are the biggest targets of fraud (Zhang and Xue, 2016; Hansen and Holroyd, 2019). Milk is considered as a healthy food, an essential component of a balanced diet from a nutritional and functional point of view, which can be used to make various dairy products and which has a positive impact on consumer’s health (Verruck et al, 2019; Hansen and Holroyd, 2019)

Objectives

Methods

Results