Journal of Food Hygiene and Safety (한국식품위생안전성학회지)

The Korean Society of Food Hygiene and Safety (한국식품위생안전성학회)
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Rapid Enumeration of Salmonella spp. in Contaminated Pork Meat Using Competitive PCR

Competitive PCR을 이용한 돼지고기 오염 살모넬라의 신속 계수

  • Published : 2007.12.31
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Abstract

In this study, the competitive polymerase chain reaction (cPCR) was used to develop a direct enumeration method of Salmonella spp. in pork meat. After comparing three DNA extraction methods, the modified guanidine thiocyanate-phenol-chloroform method was chosen for Salmonella DNA extraction in artificially inoculated pork meat. The previously reported 284-bp invA gene (Rahn et al. Mol. Cell. Probes 1992) was tested for specificity, and 57 Salmonella strains and 24 non-Salmonella strains were evaluated. All Salmonella strains tested were invA positive, and all non-Salmonella strains produced no false positive amplification products. The detection limit achieved was as low as 1,460 colony-forming units (cfu) per 0.1g of pork meat. For cPCR, the invA gene, which features a 82 bp-deletion, was cloned in the pGEM-4Z vector. A known amount of competitor DNA, which has the same primer binding sites, was co-amplified with Salmonella chromosomal DNA from the artificially inoculated pork meat. The cell-number determined by cPCR was approximately equal to the cfu from the most probable number (MPN) method. Finally, the whole procedure took only 5 hr.

돼지고기에 오염된 살모넬라의 수를 직접 측정하는 방법으로 경쟁적 PCR(competitive PCR)을 사용하였다. 세가지 DNA추출방법을 비교한 후 guanidine thiocyanate-phenol-chloroform 방법을 일부 수정하여 인위적으로 살모넬라를 오염시킨 돼지고기로부터 DNA를 직접 추출하는 방법으로 선택하였다. Rahn 등(Mol. Cell. Probes 1992년)이 이미 보고한 284-bp iuvA gene의 특이성을 평가하기 위해 살모넬라 57균주와 살모넬라가 아닌 균주 24개를 사용하였다. 시험해 본 모든 살모넬라 균주는 invA 양성으로 살모넬라가 아닌 모든 균주는 invA 음성으로 판명되었으며 살모넬라가 아닌 균주 중 양성으로 잘 못 판명된 경우는 없었다. 돼지고기 0.1 g을 사용할 경우 검출한계는 1,460 cfu였다. 경쟁적 PCR을 위해 invA gene중 82-bp를 결실시킨 DNA조각을 pGEM-4Z백터에 클로닝을 하였다. 인위적으로 오염된 돼지고기로부터 추출한 살모넬라 염색체 DNA와 이와 같은 primer결합자리를 가진 경쟁 DNA를 동시에 경쟁적 PCR로 증폭하였다. 경쟁적 PCR을 사용하여 결정한 균수와 MPN방법으로 결정한 균수는 거의 유사하였으며 전체 과정을 수행하는 데 약 5시간이 소요되었다.

Keywords

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