SPECIFIC DETECTION OF SALMONELLA SPP BY MULTIPLEX POLYMERASE CHAIN-REACTION

Three sets of oligonucleotide primers were used in the polymerase chai n reaction (PCR) assay to detect Salmonella species. phoP primers spec ific to the phoP/phoQ loci of coliform pathogenic bacteria such as Sal monella, Shigella, Escherichia coli, and Citrobacter species served as presumptive indicators of enteric bacteria. In addition to the phoP p rimers, the Hin and the H-1i primers, which targeted a 236-bp region o f hin/H2 and a 173-bp region of the H-1i flagellin gene, respectively, were used. Both Hin and H-1i primers are specific to motile Salmonell a species and are not present in Shigella, E. coli, or Citrobacter spe cies. Thus, by multiplex PCR amplification, Salmonella species includi ng Salmonella typhi, Salmonella typhimurium, Salmonella paratyphi A, a nd Salmonella enteritidis can be specifically detected. Optimal reacti on conditions have been described to demonstrate this specific, sensit ive detection of Salmonella species. By using agarose gel electrophore sis for detection of the PCR-amplified products, the sensitivity of de tection was 10(2) CFU after 25 cycles of PCR and 1 (10(0)) CFU after a 50-cycle double PCR. The efficacy of these primers was demonstrated o n environmental isolates which had previously been confirmed as Salmon ella species by the use of conventional cultural techniques. In additi on, positive amplifications resulted from Salmonella species in enviro nmental samples including soil and water.