Development of a real-time PCR assay with an internal amplification control for the screening of Shiga toxin-producing Escherichia coli in foods.

AIMS: To develop and evaluate a real-time PCR assay incorporating an internal amplification control (IAC) suitable for the screening of Shiga toxin (Stx)-producing Escherichia coli (STEC) in foods. METHODS AND RESULTS: A competitive IAC was constructed and included in an stx-specific real-time PCR assay. Coupled to 18-h enrichment and automated DNA extraction, the assay could reliably detect the presence of STEC in minced meats inoculated at 10 CFU per 25 g. Its performance was evaluated on 415 minced beef and 112 raw milk cheese samples and compared with that of a PCR-ELISA method. Fifty-three minced meats and 31 cheeses were found stx-positive, giving 98.3% and 93.75% concordance, respectively, with the PCR-ELISA reference method. CONCLUSIONS: A highly sensitive stx-specific real-time PCR method including an IAC was developed, facilitating monitoring of false-negative results due to PCR inhibitors. SIGNIFICANCE AND IMPACT OF THE STUDY: Combined with automated DNA extraction, the stx-IAC real-time PCR assay represents a suitable method for rapid screening of STEC in foods.

Development of a 5'-nuclease PCR assay for the identification of Escherichia coli strains expressing the flagellar antigen H21 and their detection in food after enrichment.

AIMS: To develop a real-time PCR assay targeting the Escherichia coli flagellar antigen H21 for identification and surveillance of clinically important Shiga toxin-producing E. coli (STEC) serotypes classified in seropathotype C. METHODS AND RESULTS: The fliC allele of STEC O91:H21 strain B2F1 was amplified and sequenced. The nucleotide sequence obtained was compared with fliC genes of E. coli O157:H21, O8:H21 and O113:H21 strains. A pair of oligonucleotide primers and a TaqMan minor groove binder probe specific for fliC-H21 were designed and used in a 5'-nuclease PCR assay. This method was evaluated using a panel of 138 diverse bacterial strains and was shown to be 100% specific for H21. PCR amplification of fliC-H21 from one cell per reaction mixture was possible, and an initial inoculum of 10 STEC H21 colony-forming units per 25 g of ground beef was detected after overnight enrichment. CONCLUSIONS: The PCR assay developed was found to be highly sensitive and specific for the identification and detection of E. coli H21 strains in ground beef. SIGNIFICANCE AND IMPACT OF THE STUDY: The real-time PCR assay targeting the H21 flagellar antigen described here offers a valuable method for the rapid detection and molecular typing of pathogenic STEC H21 strains in food.

Detection, isolation and characterization of Shiga toxin-producing Escherichia coli in retail-minced beef using PCR-based techniques, immunoassays and colony hybridization.

AIMS: To provide information on detection of Shiga toxin-producing Escherichia coli (STEC) in retail-minced beef using an approach combining (i) PCR-based techniques and automated immunoassay for stx screening and detection of the five major serogroups associated with human infection, and (ii) immunomagnetic separation (IMS) and colony hybridization assays for bacterial strain isolation. METHODS AND RESULTS: Twenty-seven out of 164 minced beef samples were stx-positive by PCR-ELISA, nine of which were also positive by real-time PCR for at least one marker of the five main serogroups tested (O26, O103, O111, O145 and O157). Two E. coli O103 stx-negative strains were isolated from two out of 10 IMS and nine STEC strains that did not belong to the five main serogroups were isolated by colony hybridization. CONCLUSIONS: PCR techniques are applicable for rapid screening of samples containing both an stx gene and an O-group marker of the five main pathogenic STEC serogroups. Isolation of STEC strains belonging to the main non-O157 serogroups remains difficult. SIGNIFICANCE AND IMPACT OF THE STUDY: This study presents an evaluation of a multi-faceted approach for the detection of the most frequently reported human pathogenic STEC serogroups. The advantages and limits of this strategy are presented.

Evaluation of a polymerase chain reaction-based test for detecting Salmonella spp. in food samples: Probelia Salmonella spp.

A commercially available polymerase chain reaction (PCR) kit was evaluated for the detection of Salmonella spp. in food samples. The test combines PCR amplification and sandwich hybridization of the amplified DNA in microtiter plates. The sensitivity and specificity was evaluated with 52 Salmonella strains and 51 non-Salmonella strains and showed that the test was entirely reliable. The threshold sensitivity was 10(2) CFU/ml. The limit of detection of dead cells that determines the minimum detection level of dead cells in food samples was superior to 10(6) CFU/25 g, a level rarely achieved in naturally contaminated samples. After an 18-h pre-enrichment step, the test could detect viable Salmonella in artificially contaminated food samples, even for the lower contamination level (3 CFU/25 g). There was complete agreement between the PCR test and the ISO 6579 bacteriological reference method with artificially contaminated samples. Regarding the accuracy of the results obtained from 253 naturally or noncontaminated foods and from 32 artificially contaminated foods, the agreement percentage was 99.6%. The fidelity of the technique was evaluated in a collaborative study with eight European laboratories and showed a correlation of 98.4%.

Off-resonance dispersion profile effect in gas laser resonators.

Diffraction losses and resonant frequencies of a gas laser resonator perturbed by an off-resonance dispersion profile, due to nonuniform radial distribution of excited atoms, are predicted by extending the ray transfer matrix formalism. These predictions are tested experimentally by using the laser beam as a probe. Experiments are done with a 6401-A He-Ne laser. In this case perturbations are induced by the strong neon absorption line at 6402 A. The two studied properties lead to determinations of the population of neon 1S5 metastable level which are both in good agreement, so the validity of the theoretical analysis presented is confirmed. It is also shown that off-resonance dispersion profile effects occur in the 6328-A He-Ne laser.

Influence of Steroidal enzyme inducers on the toxicity of pyrogallol, pargyline and nialamide.

In rats, the toxic manifestations of overdosage with with parcyline (a monoamine oxidase inhibitor) or pyrogallol (a catechol-o-methyltransferase inhibitor) were diminished by treatment with the more potent steroidal (pregnenolone-16alpha-carbonitrile, spironolactone, etc.) or nonsteroidal (phenobarbital) catatoxic substances. Except for significant protection offered by glucocorticoids (triamcinolene, prednisolone acetate) against pargyline, all other pretreatments (progesterone, estradiol, desoxycorticosterone acetate, etc.) either had no influence on or increase the deleterious effects of the two amine inhibitors. Nialamide intoxication was exacerbated by most of these conditioners.

Regulation of resistance to various toxicants by PCN (pregnenolone-16alpha-carbonitrile) and thyroxine.

In rats, PCN (the most potent catatoxic steroid known to date) at the usual dose level powerfully inhibited the toxicity of antazoline, carbamazepine, cocaine, guanethidine, ibuprofen, ketamine, LSD, nembutal and reserpine, whereas (except in the case of nembutal) thyroxine sensitized the animals to intoxication with these same compounds. Even much lower doses of PCN or thyroxine exerted similar but weaker effects. PCN-induced resistance to the various substrates was generally not altered by concurrent administration of thyroxine but in a few cases its protective action was partially or totally inhibited, depending upon the respective dose levels of both compounds.

Inhibition of the effects of alfathesin and other steroid anesthetics by catatoxic steroids in rats.

In rats, the sleeping time induced by overdosage with eight steroid anesthetics--alfathesin, 3-(3-oxo-17beta-hydroxy-19-nor-4-androsten-17alpha-yl)-propionic acid-lactone (SC-8109), 21-hydroxy=5alpha-pregnane-3,20-dione (P-234), 4-pregnene-3,11,20-trione (Bio.66), 17-hydroxy-3-oxo-4-androstene-17alpha-propionic acid-gamma-lactone(SC-5233),3alpha-hydroxy-5beta-pregnane-11,20-dione, 5beta-pregnane-3,11,20-trione (U-1373), and hydroxydione--was abolished or considerably reduced by a variety of catatoxic compounds, particularly 3beta-hydroxy-20-oxo-5-pregnene-16alpha-carbonitrile (PCN), 9alpha-fluoro-11beta,17-dihydroxy-3-oxo-4-androstene-17alpha-propionic acid potassium salt (CS-1), prednisolone, ethylestrenol and spironolactone. Phenobarbital and diphenylhydantoin, two non-steroidal stimulators of hepatic microsomal drug metabolism, were also highly effective. In contrast, triamcinolone, estradiol,progesterone, desoxycorticosterone and hydroxydione, which exert little or no catatoxic activity, failed to significantly diminish anesthesia or sedation.

3Beta-hydroxy-20-oxo-pregn-5-ene-16 alpha-carbonitrile, a potent catatoxic steroid devoid of an abortifacient effect.

PCN (3beta-Hydroxy-20-oxo-pregn-5-ene-16alpha-carbonitrile), a potent catatoxic steroid devoid of all other classic hormonal properties, given in doses amply sufficient to induce hepatic drug-metabolizing enzymes and inhibit the anesthesia caused by massive amounts of progesterone in pregnant rats, did not modify: 1. the pregnant:mated ratio, 2. the length of gestation, 3. the number of implantation sites, and 4. the size of the litter. The viability and postpartum development of the offspring were normal, as judged by gross macroscopic examination. PCN had no adverse effects on pregnancy after hypophysectomy (day 12), or ovariectomy (day 8) in rats treated with maintenance doses of progesterone and estrone. It would therefore appear that the pregnancy-maintaining capacity of endogenous or exogenous steroids (administered as substitution therapy following the glandular extirpations) was not altered by PCN and that hypothalamo-hypophyseal regulatory mechanisms were not responsible for the failure of the steroid to provoke abortion. As judged by our in vivo experiment, this pure hepatic microsomal exzyme inducer does not modify the steady state of steroid-dependent physiologic functions.