Detection and Quantification of Citrobacter freundii and C. braakii by 5′-Nuclease Polymerase Chain Reaction

Abstract

A new 5′-nuclease polymerase chain reaction (PCR) system for the detection and quantification of Citrobacter freundii and C. braakii was developed with primers and the probe oriented to a specific region of the cfa gene encoding a cyclopropane fatty acid synthase. The qualitative variant of the method consisted of a conventional PCR with end-point fluorimetry or agarose gel electrophoresis, and the quantitative variant used kinetic real-time PCR measurement. The PCR system was specific for C. freundii and C. braakii, detecting neither other Citrobacter spp. nor other enteric bacteria (Escherichia coli, Salmonella enterica, and others). The detection limit of the qualitative variant of the method was 10³ cfu/mL when the amplification was followed by fluorimetry and 10⁴ cfu/mL when the amplification was followed by gel electrophoresis. The real-time PCR variant of the method facilitated quantification over a range of concentrations from 10² to 10⁸ cfu/mL, with Escherichia coli (10⁶ cfu/mL) and Salmonella enterica (10⁶ cfu/mL) having no effect on the quantification.