| DNA Sequence analysis and rfbM gene amplification using PCR for detect salmonella C1 serogroup |
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Sung-il Lee1, Suk-chan Jung2, Jin-san Moon2, Yong-ho Park3, John-wha Lee1, Byeong-su Kim3, Byeong-kirl Baek1 |
1Bio-safety Research Institute, Chonbuk National University
2National Veterinary Research Institute, RDA
3College of Veterinary medicine, Seoul National University |
| 살모넬라 C1 serogroup 특이 rfbM 유전자 증폭과 염기서열 분석 |
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이성일1, 정석찬2, 문진산2, 박용호3, 이존화1, 김병수3, 백병걸1 |
1전북대학교 생체안전성연구소
2농촌진흥청 수의과학연구소
3서울대학교 수의과대학 |
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| Abstract |
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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. |
| Key Words:
Salmonella C1 serogroup, Salmonella cholerasuis, PCR, rfbM gene, southern blot |
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