Selective Amplification of SEA, SEB and SEC Genes by Multiplex PCR for Rapid Detection of Staphylococcus aureus

Abstract

Staphylococcus spp., is a common bacteria in cattle mastitis. Detection of the low number of bacteria requires a sensitive method. We used Multiplex Polymerase Chain Reaction (M-PCR) assay to detect the genes encoding SEA, SEB, SEC toxins that are specific in Staphylococcus aureus bacteria. The gene of 23S rRNA that is conserved in all Staphylococcus spp., also used as control. Due to the stiffness and low effective enzyme activity such as lyzozyme, proteinase k and mutilysine on bacterial cell wall, it lysed with liquid nitrogen and multiplex PCR was performed after DNA extraction. The obtained results in molecular method were compared with cell culture. The total number of 60 samples of non pasteurized milk was collected in Tehran province restrict from dairy cattle housing and 4 positive samples were detected by PCR and microbial cell culture methods. The accuracy of the test was monitored by using serial dilution (1-l0⁶) of overnight cell culture of Staphylococcus spp., bacteria (OD600:0.02 = 10⁷ cell). It showed that the sensitivity of PCR is 10 bacteria per ml of cells within few hours, meanwhile in bacterial blood agar cell culture; the number of 100 cells was detected after 48 h. This is the first report of molecular detection technique over Staphylococcus aureus bacteria in Iran which indicates the more sensitive and rapid test that can be substituted by conventional bacterial culture method.