Detection of pathogenic Escherichia coli on potentially contaminated beef carcasses using cassette PCR and conventional PCR

Abstract

Background: Over a one year period, swabs of 820 beef carcasses were tested for the presence of Shiga toxin-producing Escherichia coli by performing Polymerase Chain Reaction (PCR) in a novel technology termed "cassette PCR", in comparison to conventional liquid PCR. Cassette PCR is inexpensive and ready-to-use. The operator need only add the sample and press "go". Cassette PCR can simultaneously test multiple samples for multiple targets. Carcass swab samples were first tested for the presence of STEC genes (O157, eae, stx1 and stx2). Samples were considered to be pathogenic if positive for eae plus stx1 and/or stx2. For samples scored as pathogenic, further testing screened for 6 additional high frequency O-antigens (O26, O45, O103, O111, O121, and O145).

Results: Of the 820 samples, 41% were pathogenic and 30% were O157 positive. Of these, 19% of samples were positive for O157 and carried potentially pathogenic E. coli (eae plus stx1 and/or stx2). Of all samples identified as carrying pathogenic E. coli, 18.9, 38.8, 41.4, 0, 36.1, and 4.1% respectively were positive for O26, O45, O103, O111, O121, and O145. To validate cassette PCR testing, conventional PCR using STEC primers was performed on each of the 820 samples. Only 148 of 3280 cassette PCR tests were discordant with conventional PCR results. However, further fractional testing showed that 110 of these 148 PCRs reflected low numbers of E. coli in the enrichment broth and could be explained as due to Poisson limiting dilution of the template, affecting both cassette PCR and conventional PCR. Of the remaining 38 discordant tests, 27 initial capillary PCRs and 10 initial conventional tests were nominally discordant between cassette and conventional PCR, perhaps reflecting human/technical error on both sides of the comparison.

Conclusions: Contaminated beef carcass swabs were often complex, likely harboring more than one strain of pathogenic E. coli. Cassette PCR had 98.8% concordance with parallel conventional PCR for detection of STEC genes. This indicates that cassette PCR is highly reliable for detecting multiple pathogens in beef carcass swabs from processing plants.

Keywords: Beef carcass swabs; Cassette PCR; O-antigen complexity; STEC detection; Validation study.

Fig. 1

CCD images of a cassette with 9 trenches and 4 sets of reaction units per trench (a) at 1st PCR cycle and (b) at 35th cycle. Four columns from left to right in 9 trenches (rows) have O157, eae, stx1 and stx2 primers. The last trench (9th) has the positive control and 8th trench has the negative control. Trenches 1–7 have 7 carcass swab samples

Fig. 2

Melt curve analysis data of the cassette for: four carcass swab samples; (a) a STEC positive; (b) positive for eae and stx1 only; (c) positive for stx2 only; (d) a STEC negative; and for (e) the negative control (water); (f) the positive control of the cassette; and (g) shows agarose gel images with the conventional PCR products of the same samples used in (a), (b), (c), and (d), as well as the controls used in (e) and (f). The ladder is GeneRuler 1kb Plus DNA ladder (Themo Scientific, Carlsbad, USA)

Fig. 3

Quadruple PCRs performed in gel capillaries and in conventional PCR on three carcass swab samples. a Melt peaks of R1, R2 and R3 and (b) agarose gel image showing the ladder and the bands for PCR products. The ladder is GeneRuler 1 kb Plus DNA ladder (Themo Scientific)

Fig. 4

Reaction capillaries and an assembled cassette for cassette PCR (a) Photograph of capillaries with dried gel inside. (b) Pan with a 4 × 9 capillary array ready-to-use cassette for STEC testing