Rfbo157 by Multiplex Polymerase Chain Reaction

A rapid method for detection of Escherichia coli O157: H7 using multiplex PCR was developed. Two oligonucleotide primer pairs were used for simultaneously detection of vt encoding verotoxin genes for virulence factor and rfbO157 encoding the O-antigen specific for E. coli O157: H7. Multiplex PCR generated two products of 215 bp and 420 bp for vt and rfbO157, respectively. Multiplex PCR detected reference strain O157: H7 (NF-7777) with a sensitivity of 105 CFU per ml with no amplification of other 15 pathogenic bacteria. After incubation of 102 CFU/25 gram raw meat in tryptic soy broth at 37oC for 8 hours, multiplex PCR conducted with the addition of 100 mg bovine serum albumin produced the two specific PCR products for E. coli O157: H7. This modified multiplex PCR is a rapid, sensitive, and specific technique for detecting and differentiating E. coli O157: H7 and has the potential to be used as an alternative to conventional methods for the screening of O157: H7 strains isolated from raw meat. cases were reported with the organism as the etiological agent (Tortorello, 2000). The first report of E. coli O157: H7 outbreak was in 1982 in USA, Canada and England where this organism was found contaminating undercooked beef (Riley et al, 1983). There have been many outbreaks linked to ingestion of not only beef but also vegetables and fruits, including lettuce, cantaloupe, cabbage, alfalfa, spruots, radish sprouts and apple juice (Paton and Paton, 1998). E. coli O157: H7 possesses characteristics that are different from other E.coli. The strain O157: H7 is a slow or non sorbitol fermenter, beta-glucuronidase negative and fails to grow at temperature of 4445oC (Tortorello, 2000). Culture media including Celfixime and Tellurite Sorbitol McConkey agar (CT-SMAC) or Chrom agar O157 have been developed to suit its growth requirement. The Most Probable Number (MPN) method is used to quantify the number of organisms in liquid culture and is suitable even for small MULTIPLEX PCR FOR E. COLI O157:H7 DETECTION Vol 38 No. 1 January 2007 83 numbers (Tortorella, 2000). As biochemical tests have low sensitivity and specificity and are time consuming, a number of alternative tests with enhanced sensitivity, specificity and accuracy have been developed. Using the nucleotide sequences of vt1 and vt2, Calderwood et al (1987) employed standard PCR for the diagnosis of verotoxigenic E. coli (VTEC). Karch and Myer (1989) designed a set of primers specific to both types of vt for VTEC diagnosis. Pollard et al (1990) used 2 pairs of primers, each specific for a particular vt, in PCR base test that efficiently identified verotoxigenic E. coli in food samples. Gannon et al (1992) designed a set of primers specific to both vt genes that was effective in identifying verotoxigenic E. coli in fecal samples. Despite these available techniques, none of them has been able to specifically identify Escherichia coli O157: H7 since they are a number of other E. coli that encoded verotoxin, such as E. coli O26, O111 and O113 (Goldwater and Betteiheim, 1994). After the nucleotide sequence of rfbO157 encoding O-Ag of E. coli O157 was determined by Bilge et al (1996), test to detect E. coli O157 in milk was then developed using primers specific to rfbO157 in PCR reaction (Maurer et al, 1999). Multiplex PCR, a more specific and more rapid method employing multiple sets of primers specific for the target genes, has been employed by Fratamico et al, (1995) who used four pairs of primers specific for vt1, vt2, eaeA and EHEC-hlyA. Paton and Paton (1998), using the same set of primers, were able to detect VTEC in stool samples. However, detection via the use of 3 primer sets or more to indicate the presence of E. coli O157:H7 is not attractive due to the complexity of the reaction and length of time required. In the present study, we have developed a PCR system using 2 primers specific for vt and rfbO157 and applied it for the detection of spiked E. coli O157: H7 in meat samples, which produced a clear result. Comparative studies between PCR, immunomagnetic seperation (IMS) and direct plating technique on selective media for detecting E. coli O157: H7 were also conducted. MATERIALS AND METHODS