Multiplex PCR for Brucella identification
Bruce Ladder and Bruce Ladder suis
The Bruce Ladder (BL) and the Bruce Ladder Suis (BLS) are two molecular typing tools designed to identify Brucella species for BL (Garci-Yoldi et al., 2006 ; Lopez-Goñi et al., 2008 ; Lopez-Goñi et al., 2011) and to differentiate Brucella suis biovars, Brucella canis and Brucella microti for the BLS (Lopez-Goňi et al. 2011 ; OIE Terrestrial Manual 2018, Chapter 3.1.4). These methods are applied on DNA extracted from calibrated suspensions of isolated strains.
Principle
Both methods consist in the separation of different DNA fragment by electrophoresis previously amplified by a multiplex Polymerase Chain Reaction.
A PCR multiplex is a method allowing the amplification of several distinct DNA fragments in one reaction, with all primers related to the amplification of different loci introduced into the same reaction tube.
Primers used for the PCRs are:
Once amplified, the DNA fragments are deposited on an agarose gel and are separated according to their size by electrophoretic migration
Interpretation
After migration, a picture is taken and the DNA fragments of samples tested are compared with the fragments of reference strains (used as controls).
Real-time PCR
The Real Time Polymerase Chain Reaction (PCR) or Quantitative PCR is a molecular diagnostic tool used to detect nucleic acids, and in this case Brucella DNA (Bounaadja et al., 2009).
Principle
The present document describes a standard technique aiming at real-time Polymerase Chain Reaction (PCR) to detect the presence / absence of Brucella DNA in animal / human samples. Prior to PCR, the DNA extraction protocols have to be validated for each type of sample handled in the laboratory in order to assure standardized performances. The ID Gene® Brucella spp Triplex (IDBRU) kit is a Reel-time Polymerase Chain Reaction (Rt-PCR) kit for specific Brucella spp. sequence amplification based on TaqMan® technology. This is a qualitative Triplex system that enables simultaneous amplification of the target DNA and internal endogenous and exogenous controls.
Scientific references
HOLZAPFEL M., 2018. De l’épidémiologie moléculaire aux analyses fonctionnelles de Brucella chez les ruminants, une approche intégrée pour l’identification et l’étude de la diversité phénotypique d’un genre génétiquement homogène. Thèse de doctorat en Microbiologie, soutenue le 26-11-2018 à Paris Est, dans le cadre de l’École doctorale ABIES, 200 pages. Online document: http://www.theses.fr/2018PESC1141/document