title: Combined PCR-oligonucleotide ligation assay for rapid detection of Salmonella serovars.
abstract: We have developed a rapid and sensitive assay for the detection of Salmonella serovars in veterinary clinical specimens. This method utilizes a short cultivation period followed by PCR. For detection of the amplified product, an enzyme-linked immunosorbent assay (ELISA)-based oligonucleotide ligation assay (OLA) was used. In this study, the PCR-OLA technique was compared with conventional culture and membrane hybridization for the detection of Salmonella bacteria. In evaluating the PCR-OLA with Salmonella serovars and non-Salmonella strains of bacteria, A490 readings for 51 Salmonella strains, representing 28 serovars, were significantly higher (P < 0.05) than those for 25 non-Salmonella bacteria. With serial 10-fold dilutions of Salmonella CFU or with known concentrations of purified chromosomal DNA from Salmonella typhimurium ATCC 29946, the PCR-OLA was able to detect > or = 20 CFU per assay or > or = 80 fg of chromosomal DNA (corresponding to 160 molecules of DNA). Of 102 suspect clinical specimens screened, 15 were positive for Salmonella bacteria by both culture and the PCR-OLA procedure (100% sensitivity), and 3 samples were positive only by PCR-OLA (96.6% specificity), indicating a positive predictive value of 83.3% and a negative predictive value of 100%. In all experiments, the PCR-OLA was as sensitive as membrane hybridization. These results indicate that a limited enrichment cultivation and PCR-OLA could be used as a presumptive screening test for the detection of Salmonella serovars from any sample that currently requires extensive cultivation and that this assay would be adaptable to automation.
authors.affiliation: Department of Diagnostic Medicine & Pathobiology, College of Veterinary Medicine, Kansas State University, Manhattan 66506, USA.
authors.forename: G G
authors.initials: GG
authors.lastname: Stone
authors.forename: R D
authors.initials: RD
authors.lastname: Oberst
authors.forename: M P
authors.initials: MP
authors.lastname: Hays
authors.forename: S
authors.initials: S
authors.lastname: McVey
authors.forename: M M
authors.initials: MM
authors.lastname: Chengappa
mesh_terms: D000818:Animals; D001483:Base Sequence; D001717:Birds; D002417:Cattle; D011088:DNA Ligases; D004285:Dogs; D006736:Horses; D008969:Molecular Sequence Data; D009841:Oligonucleotides; D016133:Polymerase Chain Reaction / Q000379:methods*; D012475:Salmonella / Q000145:classification* / Q000254:growth & development; D012481:Salmonella Infections, Animal / Q000175:diagnosis* / Q000382:microbiology; D012680:Sensitivity and Specificity; D012703:Serotyping; D012756:Sheep; D013045:Species Specificity; D013552:Swine
chemical_list: D009841:Oligonucleotides; D011088:DNA Ligases