A PCR-Based assay for the detection of Escherichia coli shiga-like toxin genes in ground beef

被引:87
作者
Witham, PK
Yamashiro, CT
Livak, KJ
Batt, CA
机构
[1] CORNELL UNIV, DEPT FOOD SCI, ITHACA, NY 14853 USA
[2] PERKIN ELMER CORP, DIV APPL BIOSYST, FOSTER CITY, CA 94404 USA
关键词
D O I
10.1128/AEM.62.4.1347-1353.1996
中图分类号
Q81 [生物工程学(生物技术)]; Q93 [微生物学];
学科分类号
071005 ; 0836 ; 090102 ; 100705 ;
摘要
A detection system based on the PCR has been developed for Escherichia coli strains which harbor the Shiga-like toxin genes. This quantitative detection system involves the 5'-->3' nuclease activity of Thermus aquaticus DNA polymerase, which cleaves an internal oligonucleotide probe that has been labeled with both a fluorescent reporter dye (6-carboxy-fluorescein [FAM]) and a quencher dye (6-carboxytetramethyl-rhodamine [TAMRA]). Parameters which affected the performance of the assay included primer probe distance, probe concentration, and probe target sequence homology. The optimized assay format includes two PCR primers that generate a 497-bp amplicon specific for the sltI gene with the fluorogenic probe located 19 bp from the upstream PCR primer. When the distance between the upstream PCR primer and the probe was reduced from 190 to 19 bp, Delta RQ values increased from approximately 1.5 to 3.0. The Delta RQ for Shiga-like toxin I probe 102 reached a maximum of 4.15 at concentrations between 25 and 50 nM. The assay is sensitive and can detect approximately 10 +/- 5 CFU per PCR. As few as 0.5 CFU of Shiga-like toxin I-producing E. coli per g could be detected in ground beef with only 12 h of enrichment in modified E. coli broth.
引用
收藏
页码:1347 / 1353
页数:7
相关论文
共 43 条
  • [1] USE OF A FLUOROGENIC PROBE IN A PCR-BASED ASSAY FOR THE DETECTION OF LISTERIA-MONOCYTOGENES
    BASSLER, HA
    FLOOD, SJA
    LIVAK, KJ
    MARMARO, J
    KNORR, R
    BATT, CA
    [J]. APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 1995, 61 (10) : 3724 - 3728
  • [2] AN OUTBREAK OF ESCHERICHIA-COLI O157-H7 COLITIS ASSOCIATED WITH CONSUMPTION OF PRECOOKED MEAT PATTIES
    BELONGIA, EA
    MACDONALD, KL
    PARHAM, GL
    WHITE, KE
    KORLATH, JA
    LOBATO, MN
    STRAND, SM
    CASALE, KA
    OSTERHOLM, MT
    [J]. JOURNAL OF INFECTIOUS DISEASES, 1991, 164 (02) : 338 - 343
  • [3] AN OUTBREAK OF DIARRHEA AND HEMOLYTIC UREMIC SYNDROME FROM ESCHERICHIA-COLI O157-H7 IN FRESH-PRESSED APPLE CIDER
    BESSER, RE
    LETT, SM
    WEBER, JT
    DOYLE, MP
    BARRETT, TJ
    WELLS, JG
    GRIFFIN, PM
    [J]. JAMA-JOURNAL OF THE AMERICAN MEDICAL ASSOCIATION, 1993, 269 (17): : 2217 - 2220
  • [4] RAPID AND SIMPLE METHOD FOR PURIFICATION OF NUCLEIC-ACIDS
    BOOM, R
    SOL, CJA
    SALIMANS, MMM
    JANSEN, CL
    WERTHEIMVANDILLEN, PME
    VANDERNOORDAA, J
    [J]. JOURNAL OF CLINICAL MICROBIOLOGY, 1990, 28 (03) : 495 - 503
  • [5] UNUSUAL VEROTOXIN-PRODUCING ESCHERICHIA-COLI ASSOCIATED WITH HEMORRHAGIC COLITIS
    BOPP, CA
    GREENE, KD
    DOWNES, FP
    SOWERS, EG
    WELLS, JG
    WACHSMUTH, IK
    [J]. JOURNAL OF CLINICAL MICROBIOLOGY, 1987, 25 (08) : 1486 - 1489
  • [6] BORCZYK AA, 1987, LANCET, V1, P98
  • [7] DEVEREAUX J, 1984, GENE, V12, P729
  • [8] H7 ANTISERUM-SORBITOL FERMENTATION MEDIUM - A SINGLE TUBE SCREENING MEDIUM FOR DETECTING ESCHERICHIA-COLI O157-H7 ASSOCIATED WITH HEMORRHAGIC COLITIS
    FARMER, JJ
    DAVIS, BR
    [J]. JOURNAL OF CLINICAL MICROBIOLOGY, 1985, 22 (04) : 620 - 625
  • [9] DETECTION OF ESCHERICHIA-COLI O157/H7 BY MULTIPLEX PCR
    FRATAMICO, PM
    SACKITEY, SK
    WIEDMANN, M
    DENG, MY
    [J]. JOURNAL OF CLINICAL MICROBIOLOGY, 1995, 33 (08) : 2188 - 2191
  • [10] RAPID AND SENSITIVE METHOD FOR DETECTION OF SHIGA-LIKE TOXIN-PRODUCING ESCHERICHIA-COLI IN GROUND-BEEF USING THE POLYMERASE CHAIN-REACTION
    GANNON, VPJ
    KING, RK
    KIM, JY
    THOMAS, EJG
    [J]. APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 1992, 58 (12) : 3809 - 3815