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Improved Detection of Viable Escherichia coli O157:H7 in Milk by Using Reverse Transcriptase-PCR
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 Title & Authors
Improved Detection of Viable Escherichia coli O157:H7 in Milk by Using Reverse Transcriptase-PCR
Choi, Suk-Ho; Lee, Seung-Bae;
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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 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.
reverse transcriptase-polymerase chain reaction;Escherichia coli O157:H7;milk;heat treatment;bactericidal treatment;
 Cited by
Real Time Reverse Transcriptase-PCR to Detect Viable Enterobacteriaceae in Milk,;;

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