우유에 포함된 Salmonella enteritidis를 효과적으로 분리하는 방법을 찾고 이를 이용하여 우유속의 S. enteritidis의 량을 추정하는 방법을 개발하였다. 일정량의 S. enteritidis를 접종한 우유로부터 guanidine thiocyanate/phenol/chloroform을 이용하여 DNA를 추출한 후 중합효소반응으로 S. enteritidis 섬모항원 유전자를 선택적으로 검출함으로써 우유 1ml당 200 colony forming unit까지 검출이 가능하였고 전체 과정의 수행에 단지 5시간 정도 걸렸다. S. enteritidis 섬모항원 유전자를 cloning한 pGem-4-Sef B(-) DNA와 인위적으로 접종된 우유로부터 추출한 Salmonella DNA를 함께 중합효소반응으로 증폭한 후 제한효소로 잘라 전기영동을 행하여 band의 강도를 비교함으로써 Salmonella DNA copy수를 추정하는 것이 가능하였다.
The polymerase chain reaction was used to selectively detect sequences within the fimbrial antigen of Salmonella enteritidis. Sterile milk was artificially inoculated with known amount of S. enteritidis and then DNA was extracted with guanidine thiocyanate/phenol/chloroform, followed by PCR. A detection limit of as few as 100 colony forming unit (cfu) per 0.5 ml milk was obtained with this method. For the whole procedure, it took only 5 h. A semi-quantitative polymerase chain reaction assay which allows an estimation of colony forming unit of S. enteritidis was developed. Known amount of standard plasmid pGem-4Z-Sef B(-) containing cloned S. enteritidis fimbrial antigen gene was co-amplified with Salmonella genomic DNA isolated from artificially inoculated milk. The same set of primers were used for the amplification and the products were cleaved with Bam HI. The concentration of the target DNA could be estimated by comparing the intensity of the two bands after electrophoresis. The PCR-based protocol described in this paper provides a rapid, simple, and sensitive method for detecting S. enteritidis in milk.
