Comparison of ordinary reverse transcription real-time polymerase chain reaction (qRT-PCR) with a newly developed one-step single-tube nested real-time RT-PCR (OSN-qRT-PCR) for sensitive detection of SARS-CoV-2 in wastewater.

Wastewater monitoring has proven to be an important approach to detecting and controlling the development of the SARS-CoV-2 pandemic. Various tests based on reverse transcription real-time PCR (qRT-PCR) have been developed and used for the detection of SARS-CoV-2 in wastewater samples. In this study, we attempted to increase the sensitivity of qRT-PCR by developing a one-step single-tube nested qRT-PCR assay (OSN-qRT-PCR). Two variants were developed, oriented to nucleocapsid phosphoprotein gene (N) and to spike protein gene (S), respectively. The performance of conventional qRT-PCR assays oriented to these genes with two novel OSN-qRT-PCR assays were firstly optimized using wastewater artificially contaminated with two encapsidated RNA mimic systems harboring a portion either N or S gene (ENRM and ESRM, respectively). The assays were coupled to a polyethylene glycol-based RNA precipitation/extraction method and applied to detect SARS-CoV-2 in wastewater samples from four cities in Slovakia. Both novel OSN-qRT-PCR assays demonstrated higher detection rates than the ordinary qRT-PCR counterparts. The virus levels in the analyzed wastewater samples had a high or very high relation with the numbers of clinical cases in the monitored regions. In fact, correlation with a 3-, 4-, or 5-day temporal offset was revealed. The OSN-qRT-PCR assays demonstrated robustness, mainly in samples with low viral loads.

Useful molecular tools for facing next pandemic events: Effective sample preparation and improved RT-PCR for highly sensitive detection of SARS-CoV-2 in wastewater environment.

Viral pandemics can be inevitable in the next future. Considering SARS-CoV-2 pandemics as an example, there seems to be a need to develop a surveillance system able to monitor the presence of potential pathogenic agents. The sewage and wastewater environments demonstrated to be suitable targets for such kind of analysis. In addition, it is important to have reliable molecular diagnostic tools and also to develop a robust detection strategy. In this study, an effective sample preparation procedure was selected from four options and combined with a newly developed improved RT-PCR. First, a model viral system was constructed, containing a fragment of the SARS-CoV-2 gene encoding for the Spike protein. The encapsidated S RNA mimic (ESRM) was based on the plum pox virus (PPV) genome with the inserted targeted gene fragment. ESRM was used for seeding wastewater samples in order to evaluate the viral recovery of four different viral RNA concentration/extraction methods. The efficiency of individual approaches was assessed by the use of a quantitative reverse transcription PCR (qRT-PCR) and by a one-step single-tube nested quantitative reverse transcription PCR (OSN-qRT-PCR). For the detection of viruses in wastewater samples with low viral loads, OSN-qRT-PCR assay produced the most satisfactory results and the highest sensitivity.

Wine Yeasts Selection: Laboratory Characterization and Protocol Review.

Wine reflects the specificity of a terroir, including the native microbiota. In contrast to the use of Saccharomyces cerevisiae commercial starters, a way to maintain wines' microbial terroir identities, guaranteeing at the same time the predictability and reproducibility of the wines, is the selection of autochthonous Saccharomyces and non-Saccharomyces strains towards optimal enological characteristics for the chosen area of isolation. This field has been explored but there is a lack of a compendium covering the main methods to use. Autochthonous wine yeasts from different areas of Slovakia were identified and tested, in the form of colonies grown either on nutrient agar plates or in grape must micro-fermentations, for technological and qualitative enological characteristics. Based on the combined results, Saccharomyces cerevisiae PDA W 10, Lachancea thermotolerans 5-1-1 and Metschnikowia pulcherrima 125/14 were selected as potential wine starters. This paper, as a mixture of experimental and review contributions, provides a compendium of methods used to select autochthonous wine yeasts. Thanks to the presence of images, this compendium could guide other researchers in screening their own yeast strains for wine production.

Comparison of microbial diversity during two different wine fermentation processes.

Wine production is a complex procedure in which an important role is played by many microorganisms, particularly yeasts and bacteria. In modern wineries, alcoholic fermentation is usually carried out by adding microbial starter cultures of Saccharomyces cerevisiae strains for precisely controlled production. Nowadays, in the Slovak Republic, autochthonous vinification is getting more popular. The present article deals with the comparison of two vinification approaches, namely spontaneous fermentation and fermentation controlled by a standard commercial S. cerevisiae starter, from the point of view of microbiota dynamics and the chemical characteristics of the wines produced. The dynamics of microbial populations were determined during the fermentation process by a 16S and 28S rRNA next-generation sequencing approach. A profile of the volatile compounds during these fermentation processes was identified by solid-phase microextraction (SPME) coupled to gas chromatography-mass spectrometry (GC-MS). In summary, the microbial diversity in the m1 phase (initial must) was higher, despite the presence of the starter culture. In the m3 phase (young wine), the microbiome profiles of both batches were very similar. It seems that the crucial phase in order to study the relationship of the microbiome and the resulting product should be based on the m2 phase (fermented must), where the differences between the autochthonous and inoculated batches were more evident.

Transcription activity of lactic acid bacterial proteolysis-related genes during cheese maturation.

The catabolism of milk protein in cheese is one way how the microorganisms influence the sensorial characteristics of the final product. In this investigation, we paid attention to four genes [prtP (cell-envelope proteinase gene), pepX (X-prolyl dipeptidyl aminopeptidase gene), pepN (aminopeptidase gene) and bcaT (branched chain aminotransferase gene)] responsible for the production of volatile aroma-active compounds from milk proteins by lactic acid bacteria (LAB). We studied the dynamics of these genes and their corresponding LAB host, during the maturation of a raw ewes' milk-based cheese, using metagenomics and metatranscriptomics approaches. The transcriptome-oriented experiments included the analysis of total RNA (at three stages of cheese maturation) and also the construction of specific cDNA sub-libraries of the abovementioned genes. The proteolytic transcriptome analysis was supported by following the transcription activity of 16S rRNA gene and by metagenomic investigation. The combination of the described methods permitted to screen the dynamics of targeted genes throughout the cheese production. Lactococci were the major players in the LAB group, but the analysis provided also information on the role and properties of members of the genus Lactobacillus, such as Lb. rhamnosus, Lb. helveticus, Lb. pentosus, Lb. curvatus, Lb. parabuchneri, Lb. plantarum, Lb. brevis, Lb. delbrueckii, Lb. paracasei, Lb. fermentum and Lb. heilongjiangensis, proteolysis-related genes of which were active during cheese ripening.

Genome analysis of lactic acid bacterial strains selected as potential starters for traditional Slovakian bryndza cheese.

Genomes of 21 strains of lactic acid bacteria isolated from Slovakian traditional cheeses were sequenced on an Illumina MiSeq platform. Subsequently, they were analysed regarding taxonomic classification, presence of genes encoding defence systems, antibiotic resistance and production of biogenic amines. Thirteen strains were found to carry genes encoding at least one bacteriocin, 18 carried genes encoding at least one restriction-modification system, all strains carried 1-6 prophages and 9 strains had CRISPR-Cas systems. CRISPR-Cas type II-A was the most common, containing 0-24 spacers. Only 10% spacers were found to be homological to known bacteriophage or plasmid sequences in databases. Two Enterococcus faecium strains and a Lactococcus lactis strain carried antibiotic resistance genes. Genes encoding for ornithine decarboxylase were detected in four strains and genes encoding for agmatine deiminase were detected in four strains. Lactobacillus paraplantarum 251 L appeared to be the most interesting strain, as it contained genes encoding for two bacteriocins, a restriction-modification system, two CRISPR-Cas systems, four prophages and no genes connected with antibiotic resistance or production of biogenic amines.

Comparison of Real-Time Polymerase Chain Reaction and Enzyme-Linked Immunosorbent Assay for Sensitive and Quantitative Detection of Hazelnuts in Nut Pastes.

Background: Hazelnuts, being a frequent agent of allergenic reactions, need to be detected in food products. Thus, it is necessary to develop and further investigate appropriate methods for detection. Objective: The aim of the study was to compare the analysis of nut pastes (peanut paste spiked with different amounts of hazelnut paste) as a model of contamination of confectionery. Methods: Real-time PCR and sandwich ELISA (RidaScreen Hazelnut Fast Kit) were used. Results: For real-time PCR, LOQ of 2 mg/kg and a quantification range from 2 to 10 000 mg/kg were determined. For ELISA, LOQ of 1 mg/kg and a quantification range from 1 to 100 mg/kg were determined. Conclusions: The comparison shows that the methods had comparable sensitivity with LOQs in the same order of magnitude. Although ELISA was slightly more sensitive, it required dilution of samples at higher concentrations of the analyte because of its narrow quantification range. Results of this study suggest that real-time PCR and ELISA are both suitable methods for the analysis of nut pastes over a wide range of concentrations. Achieved results could be useful for control as well as for technological purposes. Highlights: Real-time PCR analysis of peanut paste spiked with different amounts of hazelnut paste as a model is proposed. Sandwich ELISA analysis of peanut paste spiked with different amounts of hazelnut paste as a model is proposed. The analytical parameters of real-time PCR and ELISA methods are compared.

Novel insights into microbial community dynamics during the fermentation of Central European ice wine.

Culture-dependent and culture-independent strategies were applied to investigate the microbiota of autumn undamaged and damaged berries, winter berries and ice wine must samples of Grüner Veltliner (Veltlínske zelené) from Small Carpathian wine-producing region. One hundred twenty-six yeasts and 242 bacterial strains isolated from several microbiological media (YPD, PDA, R2A, GYC, MRS and MRS-T) were clustered by ITS-PCR and subsequent Qiaxcel electrophoresis. Representatives of each cluster were identified by sequencing. The extracellular hydrolytic properties and intracellular activities of esterase and ß-glucosidase of isolates were assayed. The culture-independent approach permitted the analysis of extracted DNA and RNA coupling DGGE fingerprinting with construction of clone libraries (bacterial and fungal; DGGE-cloning). The combination of the two approaches provided comprehensive data that evidenced the presence of a complex microbiota in each analyzed sample. RNA and DNA analyses facilitated differentiation of living microorganisms from the entire microbiota. Diverse microbial communities colonized the autumn and winter berries. Generally, the combination of results obtained by the methods suggested that the must samples contained mainly Saccharomyces cerevisiae, Metschnikowia spp., Hanseniaspora uvarum, Lactococcus lactis and Leuconostoc spp. The strains exhibited interesting esterase and ß-glucosidase properties, which are important for aroma formation in wine. Fermentation strategies utilising these microorganisms, could be attempted in the future in order to modulate the ice wine characteristics.

Bacterial consortia at different wine fermentation phases of two typical Central European grape varieties: Blaufränkisch (Frankovka modrá) and Grüner Veltliner (Veltlínske zelené).

This is the first study focused to bacterial diversity and dynamic during the vinification of two important Central Europe grape vines: Blaufränkisch and Grüner Veltliner. The investigation strategy included culture-dependent and culture-independent approaches. Four different agar media were utilized for the isolation of various bacteria occurring in several fermentation stages. The isolates were clustered by fluorescent-ITS PCR and, one or more representatives of each cluster, were identified by 16 rRNA gene sequencing. The culture-independent approach, based on 16S rRNA gene amplification, combined the denaturing gradient gel electrophoresis (DGGE) method and the construction of bacterial clone library for each wine fermentation step. A complex bacterial community was identified, comprising different lactic acid bacteria and acetic acid bacteria, such as Leuconostoc spp., Lactobacillus spp. and Gluconobacter spp. Other OTUs and bacterial isolates embraced the Actinobacteria, Bacilli, Alpha-, Beta- and Gamma-proteobacteria classes. Different taxa already detected by recent studies, such as Sphingomonas, Variovorax, Pantoea, Enterobacter and Tatumella, were detected confirming the continuous occurrence of these kinds of bacteria in wine environment. Moreover, novel genera (Amycolatopsis, Hydrogenophilus, Snodgrassella, Telluria, Gilliamella, Lelliottia, and Lonsdale quercina) never detected before were recognized, too. The role of these, until now anonymous, bacteria during vinification deserves investigation, which could open a new research field in wine technology.

Tracing Staphylococcus aureus in small and medium-sized food-processing factories on the basis of molecular sub-species typing.

Contamination by Staphylococcus aureus of the production environment of three small or medium-sized food-processing factories in Slovakia was investigated on the basis of sub-species molecular identification by multiple locus variable number of tandem repeats analysis (MLVA). On the basis of MLVA profiling, bacterial isolates were assigned to 31 groups. Data from repeated samplings over a period of 3 years facilitated to draw spatial and temporal maps of the contamination routes for individual factories, as well as identification of potential persistent strains. Information obtained by MLVA typing allowed to identify sources and routes of contamination and, subsequently, will allow to optimize the technical and sanitation measures to ensure hygiene.

In silico and experimental evaluation of DNA-based detection methods for the ability to discriminate almond from other Prunus spp.

Ten published DNA-based analytical methods aiming at detecting material of almond (Prunus dulcis) were in silico evaluated for potential cross-reactivity with other stone fruits (Prunus spp.), including peach, apricot, plum, cherry, sour cherry and Sargent cherry. For most assays, the analysis of nucleotide databases suggested none or insufficient discrimination of at least some stone fruits. On the other hand, the assay targeting non-specific lipid transfer protein (Röder et al., 2011, Anal Chim Acta 685:74-83) was sufficiently discriminative, judging from nucleotide alignments. Empirical evaluation was performed for three of the published methods, one modification of a commercial kit (SureFood allergen almond) and one attempted novel method targeting thaumatin-like protein gene. Samples of leaves and kernels were used in the experiments. The empirical results were favourable for the method from Röder et al. (2011) and a modification of SureFood allergen almond kit, both showing cross-reactivity <10(-3) compared to the model almond.

Comparison of polymerase chain reaction methods and plating for analysis of enriched cultures of Listeria monocytogenes when using the ISO11290-1 method.

Analysis for Listeria monocytogenes by ISO11290-1 is time-consuming, entailing two enrichment steps and subsequent plating on agar plates, taking five days without isolate confirmation. The aim of this study was to determine if a polymerase chain reaction (PCR) assay could be used for analysis of the first and second enrichment broths, saving four or two days, respectively. In a comprehensive approach involving six European laboratories, PCR and traditional plating of both enrichment broths from the ISO11290-1 method were compared for the detection of L. monocytogenes in 872 food, raw material and processing environment samples from 13 different dairy and meat food chains. After the first and second enrichments, total DNA was extracted from the enriched cultures and analysed for the presence of L. monocytogenes DNA by PCR. DNA extraction by chaotropic solid-phase extraction (spin column-based silica) combined with real-time PCR (RTi-PCR) was required as it was shown that crude DNA extraction applying sonication lysis and boiling followed by traditional gel-based PCR resulted in fewer positive results than plating. The RTi-PCR results were compared to plating, as defined by the ISO11290-1 method. For first and second enrichments, 90% of the samples gave the same results by RTi-PCR and plating, whatever the RTi-PCR method used. For the samples that gave different results, plating was significantly more accurate for detection of positive samples than RTi-PCR from the first enrichment, but RTi-PCR detected a greater number of positive samples than plating from the second enrichment, regardless of the RTi-PCR method used. RTi-PCR was more accurate for non-food contact surface and food contact surface samples than for food and raw material samples especially from the first enrichment, probably because of sample matrix interference. Even though RTi-PCR analysis of the first enrichment showed less positive results than plating, in outbreak scenarios where a rapid result is required, RTi-PCR could be an efficient way to get a preliminary result to be then confirmed by plating. Using DNA extraction from the second enrichment broth followed by RTi-PCR was reliable and a confirmed result could be obtained in three days, as against seven days by ISO11290-1.

Principal volatile odorants and dynamics of their formation during the production of May Bryndza cheese.

Volatile aroma compounds were investigated along the production of May Bryndza cheese, a traditional Slovakian cheese produced from raw ewes' milk. Solid phase microextraction was used to isolate the volatile fractions, which were subsequently analysed by gas chromatography-olfactometry and gas chromatography-mass spectrometry. Twenty-seven key odorants were found and described in the intermediate product, ewes' curd ripened for 0, 1, 2, 4, and 8days, or in the final product. A major overall increase in the number of aroma-active volatile compounds and in their odour intensity or concentration took place during the first day of ripening of the ewes' curd, and the odour gradually culminated at the end of ripening. During the final technological step of Bryndza cheese production, when the ewes' curd ripened for 10days is decrusted, pressed and milled with NaCl solution, 8 aroma-active volatile compounds disappeared.

Microbial diversity and dynamics during the production of May bryndza cheese.

Diversity and dynamics of microbial cultures were studied during the production of May bryndza cheese, a traditional Slovak cheese produced from unpasteurized ewes' milk. Quantitative culture-based data were obtained for lactobacilli, lactococci, total mesophilic aerobic counts, coliforms, E. coli, staphylococci, coagulase-positive staphylococci, yeasts, fungi and Geotrichum spp. in ewes' milk, curd produced from it and ripened for 0 - 10 days, and in bryndza cheese produced from the curd, in three consecutive batches. Diversity of prokaryotes and eukaryotes in selected stages of the production was studied by non-culture approach based on amplification of 16S rDNA and internal transcribed spacer region, coupled to denaturing gradient gel electrophoresis and sequencing. The culture-based data demonstrated an overall trend of growth of the microbial population contributing to lactic acid production and to ripening of the cheese, lactobacilli, lactococci and Geotrichum spp. growing up to densities of 10(8) CFU/g, 10(9) CFU/g and 10(5) CFU/g, respectively, in all three consecutive batches of bryndza cheese. The diversity of bacteria encompassed Acinetobacter calcoaceticus, Acinetobacter guillouiae, Acinetobacter sp., Acinetobacter johnsonii, Citrobacter braakii, Clostridium bartlettii, Corynebacterium callunae, Corynebacterium maris, Enterobacter aerogenes, Enterobacter asburiae, Enterobacter hormaechei, Enterococcus faecium, Enterococcus pallens, Escherichia coli, Haemophilus haemolyticus, Hafnia alvei, Kluyvera cryocrescens, Lactobacillus helveticus, Lactococcus garvieae, Lc. lactis subsp. cremoris, Lc. lactis subsp. lactis, "Leuconostoc garlicum", Mannheimia glucosida, Mannheimia haemolytica, Pseudomonas sp., Ps. fluorescens, "Ps. reactans", Raoultella ornithinolytica, R. terrigena, "Rothia arfidiae", Staphylococcus aureus, Staph. epidermidis, Staph. felis, Staph. pasteuri, Staph. sciuri, Staph. xylosus, Streptococcus parauberis, Str. thermophilus and Variovorax paradoxus. The diversity of yeasts and fungi encompassed Alternaria alternata, "Ascomycete sp.", Aspergillus fumigatus, Beauveria brongniartii, Candida xylopsoci, C. inconspicua, Cladosporium cladosporioides, Debaromyces hansenii, Fomes fomentarius, Galactomyces candidus, Gymnoascus reesii, Chaetomium globosum, Kluyveromyces marxianus, Metarhizium anisopliae, Penicillium aurantiogriseum, P. camemberti, P. freii, P. polonicum, P. viridicatum, Pichia kudriavzevii, Sordaria alcina, Trichosporon lactis and Yarrowia lipolytica.

Single-tube nested real-time PCR as a new highly sensitive approach to trace hazelnut.

Hazelnut is one of the most commonly consumed tree nuts, being largely used by the food industry in a wide variety of processed foods. However, it is a source of allergens capable of inducing mild to severe allergic reactions in sensitized individuals. Hence, the development of highly sensitive methodologies for hazelnut traceability is essential. In this work, we developed a novel technique for hazelnut detection based on a single-tube nested real-time PCR system. The system presents high specificity and sensitivity, enabling a relative limit of detection of 50 mg/kg of hazelnut in wheat material and an absolute limit of detection of 0.5 pg of hazelnut DNA (1 DNA copy). Its application to processed food samples was successfully achieved, detecting trace amounts of hazelnut in chocolate down to 60 mg/kg. These results highlight the adequacy of the technique for the specific detection and semiquantitation of hazelnut as potential hidden allergens in foods.

Culture-independent detection of microorganisms in traditional Slovakian bryndza cheese.

In order to investigate the microflora of Slovakian bryndza cheese (a cheese containing unpasteurized or pasteurized ewes' milk component) by a culture-independent method, DNA was extracted directly from 7 bryndza samples and analysed by an innovative method. Using the universal prokaryotic and fungal primers, ribosomal DNA internal transcribed spacer (ITS) regions with variable length were amplified. The standard universal reverse primer L1 aligning to bacterial 23s rDNA was found unsuitable for some lactic acid bacteria and other species based on in silico analysis. Therefore, L1 primer was replaced by a combination of novel primers GplusR and GminusR aligning to the adjacent, more conserved DNA region. The amplification profiles were visualised by both standard electrophoresis and by fluorescent capillary gel electrophoresis. From representative samples, major amplicons were excised from the gel, cloned and sequenced. Sequencing revealed that the samples contained Lactobacillus delbrueckii, Lactobacillus brevis, Streptococcus thermophilus, Lactococcus lactis, Lactococcus raffinolactis, Streptococcus macedonicus, Leuconostoc pseudomesenteroides, Debaromyces hansenii, Mucor fragilis, Yarrowia lipolytica and Galactomyces geotrichum. These results represent an extension of the knowledge on the microflora of Slovakian bryndza cheese. The introduced automated ribosomal DNA intergenic spacer analysis of the bacterial and fungal genomes proved to be very effective in the application of studying microflora of cheese.

Evaluation of fungal and yeast diversity in Slovakian wine-related microbial communities.

Since the yeast flora of Slovakian enology has not previously been investigated by culture-independent methods, this approach was applied to two most common cultivars Frankovka (red wine) and Veltlin (white wine), and complemented by cultivation. Model samples included grapes, initial must, middle fermenting must and must in the end-fermentation phase. The cultured isolates were characterized by length polymorphism of rDNA spacer two region using fluorescence PCR and capillary electrophoresis (f-ITS PCR), and some were identified by sequencing. The microbial DNA extracted directly from the samples without cultivation was analysed by f-ITS PCR, amplicons were cloned and sequenced. The use of universal fungal primers led to detection of both yeasts and filamentous fungi. The amplicon of highest intensity and present in all the samples corresponded to Hanseniaspora uvarum. Other species demonstrated by both approaches included Saccharomyces sp., Metschnikowia pulcherrima or M. chrysoperlae, Candida zemplinina, Cladosporium cladosporioides, Botryotinia fuckeliana, Pichia anomala, Candida railenensis, Cryptococcus magnus, Metschnikowia viticola or Candida kofuensis, Pichia kluyveri or Pichia fermentas, Pichia membranifaciens, Aureobasidium pullulans, Alternaria alternata, Erysiphe necator, Rhodotorula glutinis, Issatchenkia terricola and Debaryomyces hansenii. Endemism of Slovakian enological yeasts was suggested on the level of minor genetic variations of the known species and probably not accounting for novel species. The prevalence of H. uvarum over Saccharomyces sp. in the samples was indicated. This is the first culture-independent study of Slovakian enology and the first time f-ITS PCR profiling was used on wine-related microbial communities.

A novel real-time polymerase chain reaction method for the detection of Brazil nuts in food.

A qualitative real-time PCR-based method for the detection of the Brazil nut (Bertholletia excelsa) component in food is described. The method consists of DNA isolation by chaotropic SPE and the subsequent PCR with Brazil nut-specific primers and a TaqMan fluorescent probe. The primers and the probe are targeted to the gene encoding for the 11s globulin of the Brazil nut. The method was positive for eight Brazil nut samples from the market and negative for all other tested plant and meat materials used in the food industry (36 samples). The intrinsic LOD of the method was 10 pg Brazil nut DNA. Using a series of model nut paste mixtures with defined Brazil nut contents, a practical detection limit of 0.1% (w/w) Brazil nut was estimated. Practical applicability of the entire method was tested by qualitative analysis of nine food samples; no discrepancies between the declared and detected Brazil nut contents were found. The presented PCR-based method is useful for sensitive and selective detection of Brazil nut in food samples.

Growth of Salmonella enterica in model mixed cultures during a two-step enrichment.

Growth of Salmonella enterica was studied in model mixed cultures with Citrobacter freundii or Escherichia coli in buffered peptone water (BPW) and in Rappaport-Vassiliadis medium with soya (RVS) with modified concentrations of MgCl2 and malachite green, and at modified incubation temperatures. Selected S. enterica strains were inoculated in BPW (10(0) cfu/ml) together with selected strains of Citrobacter freundii (up to 10(8) cfu/ml) or selected strains of Escherichia coli (up to 10(8) cfu/ml), incubated overnight and then subcultured (1: 100) in RVS variants. Growth of individual bacterial species was followed by the quantitative real-time polymerase chain reaction (PCR). Optimal culture conditions during the second selective step were: MgCl2.6 H2O concentration of 29 g/l, malachite green concentration of 36 mg/1l, and the incubation temperature of 41.5 degrees C. Citr. freundii was found to be a potent competitor and E. coli was a weaker competitor. At optimal culture conditions, competition was reduced and the density of S. enterica cultures reached the level of 10(4) cfu/ml after not later than 2 h of selective enrichment. The results obtained provide a basis for the development of a short two-step enrichment to be used in rapid real-time PCR-based methods for the detection of S. enterica in food and other matrices.