The gyrase B gene as a molecular marker to resolve interspecific relationships within the Acetobacter pasteurianus group and a novel target for species-specific PCR

Members of the Acetobacter pasteurianus are popular acetic acid bacteria (AAB) for the production of vinegar. Neither phenotypic nor the most frequently applied genotypic marker (16S ribosomal DNA) provides sufficient resolution for accurate identification of the AAB strains. In this study, the gyrB gene was used for species discrimination by direct DNA sequencing and as marker in a species-specific PCR assay. All examined A. pasteurianus strains were clearly distinguished from the closely related species by comparative sequence analysis of the gyrB gene. The average sequence similarity for the gyrB gene (82.2 %) among type strains was significantly lower than that of the 16S rRNA sequence (98.2 %). Therefore, the gyrB gene can be proposed as an additional molecular marker for A. pasteurianus and related taxa that provides higher resolution than 16S rRNA. In addition, the species-specific primers were also developed based on the gyrB and 16S rRNA gene sequences, which were then employed for PCR using the template DNA of Acetobacter strains. The PCR primer pairs were shown to be specific for A. pasteurianus, A. peroxydans and papayae. Our data indicate that the phylogenetic relationships in the A. pasteurianus group are easily resolved by direct sequencing of the gyrB gene and combined with species-specific PCR assays.     

The CYP51C gene, a reliable marker to resolve interspecific phylogenetic relationships within the Fusarium species complex and a novel target for species-specific PCR

Early diagnosis and control of different Fusarium species is essential for successful management of plant disease and subsequent prevention of toxins entering the food chain. This issue can be addressed using phylogenetic analyses and other molecular techniques, including the design of species-specific primers and corresponding PCR assays. In practice, only a few genes are sequenced for most species and insights into the evolutionary mechanisms at the species level usually stem from phylogenetic analyses of only one or a small number of genetic loci. This poses the question of whether the recovered tree accurately reflects the relationships among species or rather more local interrelationships particular to the genetic marker employed. This study examined if the Fusarium-specific CYP51C gene can be used to establish evolutionary relationships between Fusarium species and enable species-specific detection. The resolving power of the CYP51C gene was studied for 46 Fusarium isolates representing 18 different species. The resulting phylogeny analysis showed clear and well-structured separation of the isolates according to their species rank, synthesised toxin and Fusarium section. Moreover, a comparison between the individual CYP51C phylogeny and a reference tree (inferred from the concatenation of ITS, CYP51C, β-tubulin and TEF-1α sequences) indicated superior resolution of CYP51C relative to ITS and β-tubulin sequences. In addition to its suitability as a reliable marker for diagnosis of different toxigenic Fusarium species, we also show that the CYP51C gene is a promising target for development of species-specific PCR. This was demonstrated by the specific detection of Fusarium cerealis in grain samples of wheat.     

Use of PCR primers derived from a putative transcriptional regulator gene for species-specific determination of Listeria monocytogenes

Listeria monocytogenes is an opportunistic bacterial pathogen that has accounted for an important portion of human foodborne diseases worldwide. In this study, through comparative analysis of L. innocua and L. monocytogenes genomic sequences, we selected a L. monocytogenes specific gene (lmo0733) that has the potential for specific detection of L. monocytogenes. Using PCR primers (lmo0733F and lmo0733R) derived from this gene, a specific fragment of 453 bp was amplified only from genomic DNA of L. monocytogenes strains. PCR products from other Listeria species as well as other Gram-positive and -negative species were not detectable, confirming the specificity of this assay. Thus, the PCR test employing primers lmo0733F and lmo0733R represents an additional tool in the diagnostic arsenal for rapid, sensitive and specific detection and identification of human infections due to L. monocytogenes.     

Detection of tet(M) gene from raw milk by rapid DNA extraction followed by a two-step PCR with nested primers

The likelihood that milk and milk products may act as a vehicle for antibiotic-resistant bacterial genes has become a concern to the food industry and a public health issue, and the demand for rapid tests has increased. The purity of DNA extracted from food samples is a key issue in the sensitivity and usefulness of biological analyses, such as PCR for pathogens and nonpathogens. A rapid, phenol-chloroform free method based on a modification of a sodium iodide DNA extraction, followed by a two-step PCR was developed for direct detection of the tet(M) gene in milk samples within a single working day. This study compares the proposed method with a traditional phenol solvent extraction method and with a commercial kit (QIAamp DNA blood mini kit, Qiagen). The three DNA extraction methods were used to ensure access to the tet(M) gene from 1 ml of raw milk, inoculated with a strain of Enterococcus faecalis, which carries the tet(M) gene. The proposed method, followed by a two-step PCR with nested primers specific for the tet(M) gene, was able to reach a detection limit below 10 CFU/ml in less than 4 h, including the two amplification cycles, thus outperforming in sensitivity and rapidity both the traditional and the commercial method.     

Resistance of Lactobacillus casei KCTC 3260 to Reactive Oxygen Species (ROS): Role for a Metal Ion Chelating Effect

The antioxidative capacity of 4 Lactobacillus strains, isolated from a milk product, was evaluated by measuring total antioxidative ability (TAA) and resistance to reactive oxygen species (ROS). Both intact cells and intracellular cell‐free extracts of Lactobacillus casei KCTC 3260 demonstrated the highest antioxidative activity and inhibited lipid peroxidation by 46.2% and 72.9%, respectively. To evaluate the resistance of 4 Lactobacillus strains to ROS, we tested the survival under conditions of 1 mM hydrogen peroxide, 0.4 mM hydroxyl radicals, and 10 mM paraquat‐induced superoxide anions. L. casei KCTC 3260 was viable even after 8 h in the presence of 1 mM hydrogen peroxide and after 7 h in 0.4 mM hydroxyl radicals. Moreover, this strain was not influenced by superoxide anions, indicating that it has resistance to superoxide anions. To define the antioxidative mechanism, superoxide dismutase (SOD) and metal ion chelating activity were determined. All tested strains did not possess detectable SOD activity except Lactobacillus rhamnosus GG, but L. casei KCTC 3260 had the higher level of chelating activity for both Fe²⁺ and Cu²⁺ ions at 10.6 ppm and 21.8 ppm, respectively. These results suggested that the antioxidative capacity of L. casei KCTC 3260 may be caused by chelating metal ions instead of SOD activation.     

Validating allergen coding genes (Cor a 1, Cor a 8, Cor a 14) as target sequences for hazelnut detection via Real-Time PCR

Hazelnuts have been shown to contain different allergenic proteins. Amongst these, major allergens Cor a 1 and Cor a 8 and the 2S albumin Cor a 14 were selected as targets to comparatively validate three Real-Time PCR protocols. We investigated both on the choice of the amplification target, and on the matrix effect on different sample foods. Applying statistics on the validation parameters obtained from the three protocols, we showed a significant difference in Ct values. This could turn critical when a high sensitivity method is required for the detection of hazelnut traces, confirming how fundamental the choice of the template during primer design phase is. Concluding, statistical approach represents a useful tool for the identification of the best performing primer pairs in Real-Time PCR. Cor a 8 gene permitted the identification of hazelnut based ingredients in complex foods, providing a significantly higher sensitivity in the PCR amplification, when compared to Cor a 1 and Cor a 14.     

Outer membrane protein A (OmpA) may be used as a novel target to enrich and detect Escherichia coli in milk samples

In recent years, food safety incidents caused by Escherichia coli have occurred and have endangered human health. Due to the complex matrix of milk samples and the long pretreatment time, the existing methods cannot quickly detect E. coli in milk samples. It is necessary to enrich the E. coli in the complex matrix to improve the detection sensitivity. The E. coli outer membrane protein A (OmpA) is widely present on the cell membrane of E. coli and may be used as a new target to enrich E. coli. In this study, the purified recombinant OmpA protein was used to immunize BALB/c mice to produce polyclonal antibody. Immunomagnetic beads were combined with the polyclonal antibody to enrich the E. coli in the artificially contaminated milk samples. The products of immunoprecipitation were further used for PCR assay. The bacteria in the PCR sample can be pre-enriched, and the limit of detection is 10 × 10⁰ cfu/mL, which is about 100 times more sensitive than samples not processed by this method. Then, the artificially contaminated milk, coffee, juice, and soybean milk samples were tested separately, and it was found that the E. coli gene could be amplified. The whole analysis time was about 120 min, including the enrichment of bacteria and the detection of eluate. We found that OmpA combined with immunomagnetic beads was more efficient, fast, and convenient than the conventional method. Bacteria can be enriched more efficiently without extracting genomic DNA and culturing bacteria. Therefore, this method has potential value for improving the detection sensitivity and shortening the detection time of E. coli in food samples.     

A peach (Prunus persica)-specific gene, Lhcb2, used as an endogenous reference gene for qualitative and real-time quantitative PCR to detect fruit products

Among the methods used to detect food adulteration, the amplification of endogenous reference genes is particularly important. Endogenous reference genes for many different species, such as cotton, papaya, maize and others, have been reported, yet an endogenous reference gene for the peach is still lacking. In this paper, the chlorophyll a/b-binding protein (Lhcb2) gene was identified as a species-specific gene for the peach. Lhcb2 was assayed in 4 species of peaches and 8 non-peach species by both qualitative and quantitative PCR. No amplification was observed in other species. The detection limit of quantitative PCR was as low as 5 pg of DNA, equal to 9 copies, and Southern blot analysis confirmed that the Lhcb2 gene was present in a single copy in the peach genome. All of these experiments indicated that the Lhcb2 gene is a useful endogenous reference gene for the detection of peach material via both qualitative and quantitative PCR assays, even in the processed food samples such as juices containing peach.