A single-tube nested real-time polymerase chain reaction for sensitive contained detection of Cryptosporidium parvum.

AIM: A new real-time polymerase chain reaction (PCR) was developed for sensitive contained detection of Cryptosporidium parvum. METHODS AND RESULTS: The method is a nested PCR targeting a specific region of rDNA of C. parvum, which takes place in one tube, using different annealing temperatures to control the first and the second rounds of PCR, with real-time fluorogenic probe-based detection of the second round of PCR. The DNA-based detection limit of the method was 2 fg, which corresponds to approx. one genome per reaction. The detection level determined using diluted samples of C. parvum oocysts was ten oocysts per millilitre. CONCLUSIONS: The method facilitates sensitive detection of C. parvum thanks to the nested format, while reducing the risk of laboratory contamination thanks to the single-tube, real-time fluorimetric format. SIGNIFICANCE AND IMPACT OF THE STUDY: The developed method may be useful for sensitive contained detection of C. parvum in environmental and food samples, after appropriate separation of oocysts.

Identification and determination of the intra- and extracellular aminopeptidase activity by synthetic L-Ala-, L-Tyr-, and L-Phe-beta-napthylamide.

A simple, rapid and straightforward procedure for identification and determination of intracellular and extracellular activity of aminopeptidases employing synthetic substrates beta-naphtylamides of L-Ala, L-Phe, and L-Tyr was used. Poppy cells (Papaver somniferum L.) permeabilized by Tween 80 were immobilized via crosslinking by glutaraldehyde. Glutaraldehyde immobilized poppy cells lost their viability and demonstrated significantly lower aminopeptidase activities than untreated control cells probably due to a damage to the enzyme active centre. Poppy cells immobilized by pectate and alginate have retained high activity of studied aminopeptidases. The culture medium (without cells) used for the identification and determination of extracellular enzyme activities retained 20-21%, whereas intracellular activities were estimated to be 79-80% of total enzyme activity. Thus the intracellular specific activity was 1.00-1.07 higher.

Detection and quantification of Listeria monocytogenes by 5'-nuclease polymerase chain reaction targeting the actA gene.

AIMS: The aim of this study was to develop a 5'-nuclease polymerase chain reaction (PCR) for the rapid detection and quantification of Listeria monocytogenes. METHODS AND RESULTS: Specific primers and a fluorogenic probe were designed, which target a specific sequence of the actA gene encoding for a protein involved in the actin filament assembly. The PCR system was highly sensitive and specific for L. monocytogenes (inclusivity, 100%; exclusivity, 100%), which was determined using 46 L. monocytogenes and 28 non-L. monocytogenes strains. Detection limits of 10(4) cfu ml(-1) after 35 cycles and 10(2) cfu ml(-1) after 45 cycles were achieved by PCR in both real-time and end-point fluorescence measurement modes. Linear calibration lines were obtained in the range from 10(2) to 10(9) cfu ml(-1) for three L. monocytogenes strains in real-time PCR with 45 cycles. CONCLUSIONS: The developed 5'-nuclease PCR of the actA gene provides a new target for the rapid detection and quantification of L. monocytogenes. SIGNIFICANCE AND IMPACT OF THE STUDY: In conjunction with enrichment or with an appropriate quantitative sample preparation technique, the method is suitable for food safety applications.

In vivo and in vitro cloning and phenotype characterization of tellurite resistance determinant conferred by plasmid pTE53 of a clinical isolate of Escherichia coli.

A determinant encoding resistance against potassium tellurite (Te(r)) was discovered in a clinical isolate of Escherichia coli strain KL53. The strain formed typical black colonies on solid LB medium with tellurite. The determinant was located on a large conjugative plasmid designated pTE53. Electron-dense particles were observed in cells harboring pTE53 by electron microscopy. X-Ray identification analysis identified these deposits as elemental tellurium and X-ray diffraction analysis showed patterns typical of crystalline structures. Comparison with JCPDS 4-0554 (Joint Committee on Powder Diffraction Standards) reference data confirmed that these crystals were pure tellurium crystals. In common with other characterized Te(r) determinants, accumulation studies with radioactively labeled tellurite showed that reduced uptake of tellurite did not contribute to the resistance mechanism. Tellurite accumulation rates for E. coli strain AB1157 harboring pTE53 were twice higher than for the plasmid-free host strain. In addition, no efflux mechanism was detected. The potassium tellurite resistance determinant of plasmid pTE53 was cloned using both in vitro and in vivo techniques in low-copy-number vectors pACYC184 and mini-Mu derivative pPR46. Cloning of the functional Te(r) determinant into high-copy cloning vectors pTZ19R and mini-Mu derivatives pBEf and pJT2 was not successful. During in vivo cloning experiments, clones with unusual "white colony" phenotypes were found on solid LB with tellurite. All these clones were Mucts62 lysogens. Their tellurite resistance levels were in the same order as the wild type strains. Clones with the "white" phenotype had a 3.6 times lower content of tellurium than the tellurite-reducing strain. Transformation of a "white" mutant with a recombinant pACYC184 based Te(r) plasmid did not change the phenotype. However, when one clone was cured from Mucts62 the "white" phenotype reverted to the wild-type "black" phenotype. It was suggested that the "white" phenotype was the result of an insertional inactivation of an unknown chromosomal gene by Mucts62, which reduced the tellurite uptake.

Euglena gracilis as a supplementary test organism for detecting biologically active compounds.

The mutagenic activity of more than 120 antimicrobial agents and protective components was investigated. Only Kathon showed a consistent increase in revertant counts in the Ames test on Salmonella typhimurium. The hereditary bleaching test on Euglena gracilis used for detecting extranuclear mutations, showed positive results for Kathon, triethanolamine and diamine silver tetraborate.

A genotoxicological study of hexachlorobenzene and pentachloroanisole.

The potential mutagenic activity of hexachlorobenzene (HCB) and pentachloroanisole (PCA) was investigated. No genotoxicity after application on Salmonella typhimurium (Ames test), Escherichia coli, and human peripheral blood lymphocytes in vitro was observed.

A genotoxicological study of the new local anaesthetic carbamate derivatives carbisocaine, heptacaine and pentacaine.

The potential mutagenic activity of three carbamate derivatives with local anaesthetic activity was investigated. Genotoxic activity was observed after application of Carbisocaine on Euglena gracilis, whereas no activity was detected by Carbisocaine, Heptacaine and Pentacaine on Salmonella typhimurium, Escherichia coli and Drosophila melanogaster.

Hyperthermia and other factors increasing sensitivity of Euglena to mutagens and carcinogens.

The effects of different factors (temperature, light, enzymic activation) on the ability of selected mutagens and carcinogens to induce hereditary bleaching of Euglena gracilis were investigated. In the resting medium, the elevation of incubation temperature from 25 degrees C to 37 degrees C increased significantly the effect of all compounds tested on the frequency of bleached mutants of E. gracilis. The effect of light is not so unambiguous. While nitrosoguanidine (NG) exhibited practically the same bleaching activity both in the light and dark, the mutagenic effect of sodium azide (SA), nitrovin (NV), nitrosoethylurea (NEU), and benzo(a)pyrene (BP) was decreased in light. On the other hand, the light increased the bleaching activity of 5-nitro-2-furylacrylic acid (NFAA) significantly. The activation mixture S9 increased bleaching effect of NFAA and BP, whereas other mutagens were partially (NG and SA) or completely (NV and NEU) inactivated.

Mutagenic effects of two nitrofuran food preservatives.

Two structurally related food additives, 3-(5-nitro-2-furyl)acrylic acid (5-NFAA) and 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2) showed a marked mutagenic effect on Salmonella typhimurium strains TA100 and TA98 and Escherichia coli WP2 uvrA+ and WP2 uvrA. On the molar basis 5-NFAA was about two orders of magnitude less effective than AF-2. In Salmonella typhimurium TA100 anaerobic conditions stimulated the mutagenic effect of 5-NFAA which was more pronounced in nitrofuran-reductase deficient strain of Salmonella typhimurium TA100 FR50. 5-NFAA increased the number of isoleucine revertants and induced mitotic recombination at tryptophan, threonine and adenine loci of the diploid strains Saccharomyces cerevisiae a/b and Saccharomyces cerevisiae SBTD. Activity of 5-NFAA was lower than that of AF-2. The test on sex-linked recessive lethal mutations in Drosophila melanogaster indicated that only 5-NFAA is mutagenic, increasing the mutation frequency about 10-fold above the control. Results with AF-2 fell within the control range.