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Optimisation of the PCR-invA primers for the detection of Salmonella in drinking and surface waters following a pre-cultivation step

KLM Moganedi, EMA Goyvaerts, SN Venter, MM Sibara

Abstract


A polymerase chain reaction (PCR)-based method for the detection of Salmonella species in water samples was optimised and evaluated for speed, specificity and sensitivity. Optimisation of Mg2+ and primer concentrations and cycling parameters increased the sensitivity and limit of detection of PCR to 2.6 x 104 cfu/m.. A 6h non-selective pre-enrichment step further increased the limit of detection to 26 cfu/m.. Out of 14 different Salmonella strains tested, only two, Salmonella arizonae and Salmonella pullorum, did not give positive amplification results with primers homologous to a conserved region of the invA gene. When environmental and drinking waters were assessed, a non-selective pre-enrichment step was included to increase
the detection efficiency of PCR. The PCR method demonstrated specificity in the presence of other competing micro-organisms
as confirmed by the conventional culture method. No false positives or negatives were observed when household and environmental water samples were tested by invA-PCR analysis parallel to the culture method.



http://dx.doi.org/10.4314/wsa.v33i2.49060
AJOL African Journals Online