Improved Detection of Viable Escherichia coli O157:H7 in Milk by Using Reverse Transcriptase-PCR

• 발행기관: 한국축산식품학회
• 저자명: Suk Ho Choi Seung Bae Lee
• 간행물 정보: 『Food Science of Animal Resources』Korean Journal for Food Science of Animal Resources 제31권 제2호, 158~165쪽, 전체 8쪽
• 주제분류: 농수해양 > 식품과학
• 파일형태: PDF
• 발행일자: 2011.04.30

영문 초록

A sensitive reverse transcriptase-PCR (RT-PCR) method to detect viable Escherichia coli O157:H7 in milk was established. The primer sets were designed based on the nucleotide sequences of the rfbE (per) and wbdN genes in the O157 antigen gene cluster of E. coli O157:H7. RT-PCR using five different primer sets yielded DNA with sizes of 655, 518, 450, and 149-bp, respectively. All five of the E. coli O157:H7 strains were detected by RT-PCR, but 11 other bacterial species were not. The sensitivity of RT-PCR was improved by adding yeast tRNA as a carrier to the crude RNA extract. The RTPCR amplifying the 149-bp DNA fragment was the most sensitive for detecting E. coli O157:H7 and the most refractory to the bactericidal treatments. Heat treatment at 65℃ for 30 min was the least inhibitory of all bactericidal treatments. Treatment with RNase A strongly inhibited the RT-PCR of heated milk but not unheated milk. This study described RT-PCR methods that are specific and sensitive with a detection limit of 10 E. coli O157:H7 cells, and showed that pre-treating milk samples with RNase A improved the specificity to detect viable bacteria by RT-PCR.

키워드

reverse transcriptase-polymerase chain reaction Escherichia coli O157:H7 milk heat treatment bactericidal treatment