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

被引:1
作者
Choi, Suk Ho [1 ]
Lee, Seung-Bae [1 ]
机构
[1] Sangji Univ, Div Anim Resources & Life Sci, Wonju 220702, South Korea
关键词
reverse transcriptase-polymerase chain reaction; Escherichia coli O157:H7; milk; heat treatment; bactericidal treatment; POLYMERASE-CHAIN-REACTION; LISTERIA-MONOCYTOGENES; MULTIPLEX PCR; REACTION ASSAY; O157-H7; O157H7; GENES; RNA; PERSISTENCE; SAMPLES;
D O I
10.5851/kosfa.2011.31.2.158
中图分类号
TS2 [食品工业];
学科分类号
0832 ;
摘要
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 RT-PCR 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 degrees C 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.
引用
收藏
页码:158 / 165
页数:8
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