DNA Sequence analysis and rfbM gene amplification using PCR for detect salmonella C1 serogroup

살모넬라 C1 serogroup 특이 rfbM 유전자 증폭과 염기서열 분석

  • Lee, Sung-il (Bio-safety Research Institute, Chonbuk National University) ;
  • Jung, Suk-chan (National Veterinary Research Institute, RDA) ;
  • Moon, Jin-san (National Veterinary Research Institute, RDA) ;
  • Park, Yong-ho (College of Veterinary medicine, Seoul National University) ;
  • Lee, John-wha (Bio-safety Research Institute, Chonbuk National University) ;
  • Kim, Byeong-su (College of Veterinary medicine, Seoul National University) ;
  • Baek, Byeong-kirl (Bio-safety Research Institute, Chonbuk National University)
  • 이성일 (전북대학교 생체안전성연구소) ;
  • 정석찬 (농촌진흥청 수의과학연구소) ;
  • 문진산 (농촌진흥청 수의과학연구소) ;
  • 박용호 (서울대학교 수의과대학) ;
  • 이존화 (전북대학교 생체안전성연구소) ;
  • 김병수 (군산전문대) ;
  • 백병걸 (전북대학교 생체안전성연구소)
  • Received : 1995.09.14
  • Published : 1996.03.25

Abstract

The Salmonella rfb gene encoding for the biosynthesis of the oligosaccharide-repeating units of the O-antigenic determinants was cloned and sequenced. A set of nucleotide primers(a forward and reverse) was selected to target a defined region of the guanosine diphospho-mannose(GDP-Man) pyrophosphorylase synthase gene : rfbM of Salmonella C serogroup. The primer set was used to develop a PCR-based rapid and specific detection system for Salmonella C1 serogroup. Amplification bands of predicted size(1,422bp) were generated from 11 different Salmonella C1 isolates. The bands were verified to be specific for the C1 serogroup by Southern blot analysis using reference homologous DNA specificity was further confirmed by the lack of reactivity with heterologous DNA derived from non-salmonella members of the family enterobacteriaeceae. A specificity of 100% was deduced along with a very high sensitivity shown by a detection limit of 1fg of a purified DNA template. The isolated DNA sequence was found to be 99.8% homologous to S montevideo but the related primers amplified with the predicted band sizes with all the Salmonella C1 serogroups tested. It is concluded that the PCR protocol based on the rfbM gene from S cholerasuis is optimal fast and specific for the detection of Salmonella C1 serogroup and also the corresponding probe is suitable for rapid detection of all Salmonella C1 serogroup DNA tested. This technology should facilitate the identification of contaminated pig products and for any other products contaminated with the Salmonalla C1 serogroup. The immediate impact of this developed method will be in the area of food safety of pig products with the potential prospect for adaptation to other food inspection technologies.