우유 내 활력있는 Salmonella를 검출하기 위한 fimA 유전자의 역전사중합효소연쇄반응의 개발
Development of Reverse Transcriptase-Polymerase Chain Reaction of fimA Gene to Detect Viable Salmonella in Milk
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초록
살균된 우유에 오염된 Salmonella의 신속한 검출 방법을 공중 보건의 보호를 위하여 중요하다. RT-PCR 방법은 생균과 사균을 분별하여 검출할 수 있는 분자유전학적 방법이다. 본 연구의 RT-PCR 방법은 Salmonella의 type 1 fimbriae의 단량체 단백질을 암호화하는 fimA 유전자의 mRNA를 주형으로 하여 DNA를 증폭하는 방법으로서 활력이 있는 Salmonella를 검출할 수 있기 위하여 개발되었다. RNA 시료를 RQ RNase-free DNase로 처리하여 오염된 DNA를 파괴하여 RT-PCR 반응에서 주형으로서 DNA가 합성되는 것을 방지할 수 있었다. 이 RT- PCR 방법은 Salmonella 7균주를 검출하였으나 Escherichia coli, Shigella sonnei, Citrobacter freundii, 및 Klebsiella pneumoniae 는 검출하지 않았다. 우유 내에서 열처리된 107/ml과 106/ml 세균수의 Salmonella는 검출되었으나 105~100/ ml는 검출되지 않았다. RT-PCR를 검출할 수 있는 활력이 있는 Salmonella의 최소 세균수는 100/ml 이었다.
Abstract
Rapid detection of viable Salmonella in pasteurized milk is important to protect public health from food poisoning. Reverse transcriptase-polymerase chain reaction(RT-PCR) is recognized as a molecular genetical method to differentiate between live and dead bacteria. The RT-PCR in this study was designed to detect specifically viable Salmonella in milk by using the primers whose nucleotide sequences were determined based on fimA gene which encodes the subunit of type 1 fimbriae. Treatment of RNA preparation with RNase-free DNase was adequate enough to destroy DNA, which may otherwise be amplified in the RT-PCR. Seven strains of Salmonella were detected in the RT-PCR but Escherichia coli, Shigella sonnei, Citrobacter freundii, and Klebsiella pneumoniae were not. 107/ml and 106/ml of dead Salmonella which were heat-treated in milk were detectable by using the RT-PCR but 105~10/ml of the dead bacteria were not. The sensitivity of the RT-PCR in detecting viable Salmonella was 100 cells/ml.
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