PCR Detection of the Bacteroides fragilis Enterotoxin Gene Relies on Robust Primer Design.

Colonic carriage of enterotoxigenic Bacteroides fragilis (ETBF) is reportedly more common in people presenting with colorectal cancer (CRC) than in healthy controls (1–3), fueling speculation that persistent carriage of these bacteria may “drive” colon carcinogenesis (4). Our aim was to use a bft amplification protocol to determine the prevalence of toxin-producing strains of B. fragilis in CRC patient stool samples. Sixty-one patients histologically diagnosed with CRC consented to provide a stool sample prior to surgery. The study was approved by the Upper South A Regional Ethics Committee. Each stool was cultured anaerobically on Bacteroides bile esculin (BBE) agar (Fort Richard Laboratories, Auckland, New Zealand). After 48 h of incubation at 37°C, all colonies were swept off the plates into 500 l of sterile water and heated for 10 min at 99°C. Each sample was screened for evidence of the bft gene using Pantosti (5) and Odamaki (6) primer sets. The PCR mixtures for the Pantosti toxin primers consisted of a 10l volume containing 5 pmol of each primer, 200 nM deoxynucleoside triphosphates (dNTPs), 2 mM MgCl2, 1 enzyme buffer, 0.5 U HotFire polymerase (Solis Biodyne, Tartu, Estonia), and 0.5 l DNA template. The PCR mixture for amplification of the toxin gene by Odamaki primers differed by using 1.5 mM MgCl2 and by the addition of 1 l of S solution (Solis Biodyne, Tartu, Estonia). Amplification consisted of a 15-min incubation at 95°C followed by 35 cycles of 30 s of denaturation at 95°C, 30 s of annealing (52°C and 66°C, respectively), and 30 s of extension at 72°C, followed by a final extension step of 2 min at 72°C. Positive and no-template controls were included in each experiment. Products were run on a 1.5% agarose gel, and fragments were visualized using SYBRSafe dye (Invitrogen, Carlsbad, CA, USA). Results showed that the Pantosti primers amplified only 5 ETBF samples while the Odamaki primers amplified 10. Only four samples amplified by Odamaki primers were recognized by the Pantosti primers, while one sample amplified by the Pantosti primers was not recognized by the Odamaki primers (Table 1). ETBF-positive samples were further analyzed by subtype-specific PCR (6). Notably, the Pantosti primers gave no discernible product other than BFT-1, whereas samples that yielded positive bft fragments with Odamaki primers only were identified as containing BFT-2 and -3 subtypes. This finding was confirmed using both primer sets to amplify DNA extracted from three ETBF reference strains (strains VPI 13784 [BFT-1] [7], 86-5443-2-2 [BFT-2] [8], and Korea 570 [BFT-3] [9], generously supplied by Cynthia Sears). The Odamaki primer set amplified all three subtypes well, whereas the Pantosti primers amplified less efficiently from BFT-2 and very poorly from BFT-3 (Fig. 1). Collectively, these results suggest that Pantosti primers may miss some BFT-2and BFT-3containing samples. The Pantosti primer set, still in common use (3, 10), was designed against the sequence published by Moncrief and col-