Allele-specific PCR method based on rfbS sequence for distinguishing Salmonella gallinarum from Salmonella pullorum: serotype-specific rfbS sequence polymorphism.
Cloning and sequence analysis of rfbS gene identified two polymorphic nucleotides, one at position 598 (Salmonella gallinarum-specific) and other at position 237 (Salmonella pullorum-specific). Based on S. gallinarum-specific nucleotide found at position 598, an allele-specific PCR method was developed for serotype-specific detection of S. gallinarum. This PCR method was able to discriminate pure cultures of S. gallinarum from S. pullorum and other Salmonella serotypes from serogroup D in less than 3 h. Serotype-specific detection of S. gallinarum was possible in less than 24 h when the PCR was applied on the presumptive Salmonella colonies obtained after overnight incubation of selective media plates streaked with the clinical material from diseased chickens. As little as 100 pg of genomic DNA could be detected with S. gallinarum-specific primers; no PCR product was detected in non-S. gallinarum serotypes of serogroup D and other closely related non-salmonella organisms. This rfbS allele-specific amplification assay is specific, reproducible and less time consuming than the standard bacteriological methods used to detect S. gallinarum and could be an effective molecular tool for rapid definitive diagnosis of fowl typhoid in the areas of endemicity where fowl typhoid infection exists.
Shah
Devendra H
DH
Biosafety Research Institute, Department of Veterinary Internal Medicine, College of Veterinary Medicine, Chonbuk National University, Jeonbuk, Jeonju 561-756, South Korea.
Park
Jin-Ho
JH
Cho
Mae-Rim
MR
Kim
Myeong-Cheol
MC
Chae
Joon-Seok
JS
D000483:Alleles; D000818:Animals; D001483:Base Sequence; D002238:Carbohydrate Epimerases / Q000235:genetics*; D002645:Chickens; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004196:Disease Outbreaks / Q000662:veterinary*; D007723:Korea / Q000453:epidemiology; D008969:Molecular Sequence Data; D019081:O Antigens / Q000737:chemistry / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000662:veterinary; D011110:Polymorphism, Genetic; D011201:Poultry Diseases / Q000175:diagnosis / Q000453:epidemiology / Q000382:microbiology*; D015203:Reproducibility of Results; D012475:Salmonella / Q000201:enzymology / Q000235:genetics*; D012481:Salmonella Infections, Animal / Q000175:diagnosis / Q000453:epidemiology / Q000382:microbiology*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA
D004269:DNA, Bacterial; D019081:O Antigens; D002238:Carbohydrate Epimerases; C061271:paratose synthase