RAPID IDENTIFICATION OF SALMONELLA SEROVARS IN FECES BY SPECIFIC DETECTION OF VIRULENCE GENES, INVA AND SPVC, BY AN ENRICHMENT BROTH CULTURE-MULTIPLEX PCR COMBINATION ASSAY

In order to make a rapid and definite diagnosis of Salmonella enteriti s in children, an enrichment broth culture-multiplex PCR combination a ssay was devised to identify Salmonella serovars directly from fecal s amples, Two pairs of oligonucleotide primers were prepared according t o the sequences of the chromosomal invA and plasmid spvC genes, PCR wi th these two primers would produce either one amplicon (from the invA gene) or two amplicons (from the invA and spvC genes), depending on wh ether or not the Salmonella bacteria contained a virulence plasmid, Th e fecal sample was diluted 10- to 20-fold into gram-negative enrichmen t broth and incubated to eliminate inhibitory compounds and also to al low selective enrichment of the bacteria, One or two amplicons were ob tained, the expected result if Salmonella bacteria were present, The d etection limit of this PCR was about 200 bacteria per reaction mixture . The primers were specific, as no amplification products were obtaine d with 18 species and 22 isolates of non-Salmonella bacteria tested wh ich could be present in the feces or cause contamination. In contrast, when 23 commonly seen Salmonella serovars (38 isolates) were tested, all were shown to carry the invA gene and seven concomitantly harbored the spvC gene of the virulence plasmid, This assay was applied to the diagnosis of Salmonella enteritis in 57 children who were suffering f rom mucoid and/or bloody diarrhea, Of the 57 children, 38 were PCR pos itive and 22 were culture positive, There were two culture-positive sa mples that were not detected by PCR. Thus, this PCR assay showed an ef ficiency of 95% (38 of 40), which is much higher than the 60% (24 of 4 0) by culture alone. Not only is this method more sensitive, rapid, an d efficient but it will cause only an incremental increase in the cost of stool processing, since enrichment cultivation of fecal samples fr om diarrheal patients using gram-negative enrichment broth is a routin e practice for identification in many diagnostic microbiology laborato ries, This PCR method, therefore, has clinical application.