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Given the recognized nutritional value of fish and shifting consumer lifestyles, processed seafood has become increasingly prevalent, comprising a significant portion of global food production. Although current European Union labeling regulations do not require species declaration for these products, food business operators often voluntarily provide this information on ingredient lists. Next Generation Sequencing (NGS) approaches are currently the most effective methods for verifying the accuracy of species declarations on processed seafood labels. This study examined the species composition of 20 processed seafood products, each labeled as containing a single species, using two DNA metabarcoding markers targeting the mitochondrial cytochrome c oxidase I (COI) and 16S rRNA genes. The combined use of these markers revealed that the majority of the products contained multiple species. Furthermore, two products were found to be mislabeled, as the declared species were not detected. These findings underscore that NGS is a robust technique that could be adopted to support routine food industry activities and official control programs, thereby enhancing the 'From Boat to Plate' strategy and combating fraudulent practices in the complex fisheries supply chain. [ABSTRACT FROM AUTHOR]

We evaluated the specificity of the primers OPF-01, P54, and 1253 to identify A. fumigatus, A. flavus, and A. niger, respectively, with the RAPD-PCR method. Eighty-two isolates belonging to the sections Fumigati, Flavi, and Nigri were used. The isolates were identified by phenotypic (macro- and micromorphology) and genotypic (partial sequences of the BenA gene) methods. The RAPD-PCR method was used to obtain polymorphic patterns with the primers OPF-01, P54, and 1253. The specificity of the polymorphic patterns of the isolates of each species was evaluated through the UPGMA clustering method and logistic regression model. All isolates of the genus Aspergillus were identified at the section level by macro- and micromorphology showing the typical morphology of the sections Fumigati, Flavi, and Nigri, and the species were identified by the construction of the phylogeny of the partial sequence of the BenA gene. The patterns' polymorphic strains obtained with the primers OPF-01, P54, and 1253 for the isolates of A. fumigatus, A. flavus, and A niger, respectively, showed the same polymorphic pattern as the reference strains for each species. To verify the specificity of the primers, they were tested with other species from the sections Fumigati, Flavi and Nigri. The results support that the primers OPF-01, P54, and 1253 generate polymorphic patterns by RAPD-PCR species specific to A. fumigatus, A. flavus, and A. niger, respectively. [ABSTRACT FROM AUTHOR]

Simple Summary: A lot of avian species are morphologically monomorphic, and thus, sex determination is difficult if it is based only on their external examination. Molecular sex identification has become a crucial tool in various fields of biology, including ornithology. This non-invasive method used to determine the genetic sex of birds of any age, in our case, Eudromia elegans (Paleognathae, Tinamiformes), can proceed with regard to the differences between avian male (ZZ) and female (ZW) chromosomes. According to our experience, the other molecular methods failed in the sex determination of this exact species. The new test was designed to be reliable, fast, and simple to determine the sex of the individuals of E. elegans. Molecular sexing has significant implications for understanding various aspects of its biology, including mating systems, population genetics, and conservation strategies. By accurately determining the sex of individuals within a population, researchers can investigate sex ratio patterns, reproductive success, and gene flow. Sex determination based just on morphological traits such as plumage dichromatism, sexual size dimorphism, behavior, or vocalizations is really challenging because of the sexual monomorphism present in more than half of avian species. Currently, a lot of them can be tested through DNA-based procedures, but they do not fit all the avian species, such as Eudromia elegans. The aim of this study was to design a new molecular method suitable for routine sex determination for that species that is fast, simple, and cost- and time- effective. DNA was isolated from dry blood stain and/or chest feather samples of E. elegans species. We used two sets of sex-specific primers (ZF/ZR and WF/WR) to amplify the expected fragments localized on the highly conserved CHD1 gene to distinguish between sexes due to the W-specific DNA sequence present only in females. We confirmed the accuracy and consistency of the PCR-based method based on length differences to distinguish between the sexes of E. elegans, which amplified two fragments in females and one fragment in males. [ABSTRACT FROM AUTHOR]

The loop-mediated isothermal amplification (LAMP) technique is a great alternative to PCR-based methods, as it is fast, easy to use and works with high sensitivity and specificity without the need for expensive instruments. However, one of the limitations of LAMP is difficulty in achieving the simultaneous detection of several targets in a single tube, as the methodologies that allow this rely on fluorogenic probes containing specific target sequences, complicating their adaptation and the optimization of assays. Here, we summarize different methods for the development of multiplex LAMP assays based on sequence-specific detection, illustrated with a schematic representation of the technique, and evaluate their practical application based on the real-time detection and quantification of results, the possibility to visualize the results at a glance, the prior stabilization of reaction components, promoting the point-of-care use, the maximum number of specific targets amplified, and the validation of the technique in clinical samples. The various LAMP multiplexing methodologies differ in their operating conditions and mechanism. Each methodology has its advantages and disadvantages, and the choice among them will depend on specific application interests. [ABSTRACT FROM AUTHOR]

Inherited retinal diseases (IRDs) are a clinically and genetically heterogeneous group of diseases which cause visual loss due to Mendelian mutations in over 250 genes, making genetic diagnosis challenging and time-consuming. Here, we developed a new tool, CDIP (Cost-effective Deep-sequencing IRD Panel) in which a simultaneous sequencing of common mutations is performed. CDIP is based on simultaneous amplification of 47 amplicons harboring common mutations followed by next-generation sequencing (NGS). Following five rounds of calibration of NGS-based steps, CDIP was used in 740 IRD samples. The analysis revealed 151 mutations in 131 index cases. In 54 (7%) of these cases, CDIP identified the genetic cause of disease (the remaining were single-heterozygous recessive mutations). These include a patient that was clinically diagnosed with retinoschisis and found to be homozygous for NR2E3-c.932G>A (p.R311Q), and a patient with RP who is hemizygous for an RPGR variant, c.292C>A (p.H98N), which was not included in the analysis but is located in proximity to one of these mutations. CDIP is a cost-effective deep sequencing panel for simultaneous detection of common founder mutations. This protocol can be implemented for additional populations as well as additional inherited diseases, and mainly in populations with strong founder effects. [ABSTRACT FROM AUTHOR]

In this study, the primers of different protocols for the detection of White Spot Syndrome Virus (WSSV) were analyzed in silico to evaluate their potential performance in PCR. As with any biological entity, this virus evolves constantly. Thus, this analysis showed that a few primers, including those recommended by the World Organization for Animal Health (WOAH), might mismatch with some isolates of WSSV, specially with isolates more recently sequenced. Furthermore, a set of primers recommended by WOAH, showed the potential to self-dimer and form hairpin loop structures, which could affect the efficiency of PCR, resulting in an inaccurate diagnostic result. From our perspective, and considering the evolutionary trajectory of this virus, it may be time for the WOAH to update the PCR protocols recommended for WSSV detection, which remains as a highly prevalent and lethal virus. [ABSTRACT FROM AUTHOR]

Red algae (Rhodophyta) are a heterogeneous group of marine algal species that have served as a source of high-value molecules, including antioxidants and scaffolds, for novel drug development. However, it is challenging to identify Rhodophytes through morphological features alone, and in most instances, that has been the prevailing approach to identification. Consequently, this study undertook the identification of red algae species in Kenton-on-Sea, South Africa, as a baseline for future research on red algae biodiversity and conservation. The identification was achieved by designing, analysing, and using a set of universal primers through DNA barcoding of the rbcL gene. The PCR products of the rbcL gene were sequenced, and 96% of the amplicons were successfully sequenced from this set and matched with sequences on BOLD, which led to these species being molecularly described. Amongst these species are medicinally essential species, such as Laurencia natalensis and Hypnea spinella, and potential cryptic species. This calls for further investigation into the biodiversity of the studied region. Meanwhile, the availability of these primers will ease the identification process of red algae species from other coastal regions. [ABSTRACT FROM AUTHOR]

Acetic acid bacteria (AAB) are ubiquitous wine spoilage microorganisms causing significant economic damage to winemakers. Considering difficulties in their isolation through traditional microbiological methods, it would be advantageous to detect them using molecular methods at all stages of winemaking and, thus, prevent wine spoilage. In this research, we analyzed wines, musts and grapes of 13 varieties grown in different regions of the Republic of Moldova. The DNA was extracted and analyzed via PCR using home-designed primers to detect Acetobacter aceti and Acetobacter pasteurianus. Generally, samples with no detectable amounts of AAB in either musts or wine had volatile acidity within the acceptable limits. Only one grape (Rara Neagra) had detectable amounts of AAB (A. pasteurianus) at all analyzed stages (grape, must, wine), and this sample had the highest amount of volatile acidity (2.11 g/L), exceeding the maximum acceptable limit for red wines of 1.2 g/L. A. pasteurianus was more common than A. aceti, both in musts and wines. Samples positive for AAB but containing low amounts of them in wine (Cq value > 35) did not have volatile acidity above the acceptable level. Samples that were wine-negative but must-positive for AAB had volatile acidity close to the acceptable limit. This study shows the utility of PCR diagnostics for predicting the risks of wine spoilage by AAB. [ABSTRACT FROM AUTHOR]

The application of organic coatings is a common way of protecting metal substrates against corrosion. To dry the coating faster, catalytic infrared radiation (IR) can be applied. This paper aims to assess the differences in the physical, chemical, and corrosion properties of primer coatings dried with catalytic infrared radiation, compared to the same coatings dried under atmospheric conditions. Corrosion properties were characterized using humidity and a salt spray chamber, as well as electrochemical impedance spectroscopy (EIS), preceded by open circuit potential (OCP) measurement. Pencil hardness, cross-cut, and pull-off adhesion tests were used to compare the properties of examined primers before and after testing in the corrosion acceleration chambers. The microstructure and distribution of chemical composition were studied by scanning electron microscope (SEM) with energy-dispersive X-ray spectroscopy (EDX) together with Fourier-transform infrared spectroscopy (FTIR). Phase transitions in the coating were determined by differential scanning calorimeter (DSC). Infrared-dried primers achieved a higher curing degree. Therefore, their mechanical and corrosion properties are superior when compared to the same coatings dried under atmospheric conditions. [ABSTRACT FROM AUTHOR]

Due to its unique properties, zirconia is increasingly being used in dentistry, but surface preparation for bonding is difficult because of its polycrystalline structure. This study aimed to determine the effect of a new etching technique (Zircos-E) on Ceramill Zi (Amann Girrbach). The effect of etching and the use of primers (Monobond Plus and MKZ Primer) on the bond strength of zirconia with resin cement (NX3) was assessed. Shear bond strength was evaluated after storage in water for 24 h and after thermal aging (5000 thermocycling at 5 °C/55 °C). A scanning electron microscope (Hitachi S-4700) was used to evaluate the surface structure before and after the Zircos-E system. The roughness parameters were assessed using an SJ-410 profilometer. The etched zirconia surface is more homogeneous over the entire surface, but some localized forms of erosion exist. The etching of zirconia ceramics caused changes in the surface structure of zirconia and a significant increase in the shear bond strength between zirconia and resin cement. The use of primers positively affects the adhesion between resin cement and zirconia. Aging with thermocycler significantly reduced the shear bond strength, with one exception—sandblasted samples with MKZ Primer. Standard ceramic surface preparation, involving only alumina sandblasting, does not provide a satisfactory bond. The use of etching with the Zircos-E system and primers had a positive effect on the strength of the zirconium–resin cement connection. [ABSTRACT FROM AUTHOR]

The aim of the study was to evaluate particular polymerase chain reaction primers targeting selected representative genes and the influence of a preincubation step in a selective broth on the sensitivity of group B Streptococcus (GBS) detection by nucleic acid amplification techniques (NAAT). Research samples were vaginal and rectal swabs collected in duplicate from 97 pregnant women. They were used for enrichment broth culture-based diagnostics, bacterial DNA isolation, and amplification, using primers based on species-specific 16S rRNA, atr and cfb genes. To assess the sensitivity of GBS detection, additional isolation of samples preincubated in Todd-Hewitt broth with colistin and nalidixic acid was performed and then subjected to amplification again. The introduction of the preincubation step increased the sensitivity of GBS detection by about 33–63%. Moreover, NAAT made it possible to identify GBS DNA in an additional six samples that were negative in culture. The highest number of true positive results compared to the culture was obtained with the atr gene primers, as compared to cfb and 16S rRNA primers. Isolation of bacterial DNA after preincubation in enrichment broth significantly increases the sensitivity of NAAT-based methods applied for the detection of GBS from vaginal and rectal swabs. In the case of the cfb gene, the use of an additional gene to ensure the appropriate results should be considered. [ABSTRACT FROM AUTHOR]

Rapid and modular assembly of DNA parts is crucial to many synthetic biologists. This can be achieved through Golden Gate assembly, which often requires purchase and delivery of new primers for each part and assembly configuration. Here, we report on a small set of primers that can be used to amplify any DNA from the Registry of Standard Biological Parts for Golden Gate assembly. These primers bind to regions common to the backbone plasmid for these parts, but pair imperfectly and introduce type IIS restriction enzyme sites in a way that minimizes assembly scars. This approach makes redesign of assembly strategies faster and less expensive and can help expand access to synthetic biology to a wider group of scientists and students. [ABSTRACT FROM AUTHOR]

Listeria monocytogenes is a major human foodborne pathogen responsible for listeriosis. The virulence factor Internalin A (inlA) has a key role in the invasion of L. monocytogenes into the human intestinal epithelium, and the presence of premature stop-codons (PMSC) mutations in the inlA gene sequence is correlated with attenuated virulence. The inlA sequencing process is carried out by dividing the gene into three sections which are then reassembled to obtain the full gene. The primers available however were only able to entirely amplify the lineage II isolates. In this study, we present a set of new primers which allow inlA sequencing of isolates belonging to both lineages, since lineage I isolates are the ones most frequently associated to clinical cases. Using newly designed primers, we assessed the presence of inlA PMSCs in food, food processing environments and clinical isolates. [ABSTRACT FROM AUTHOR]

This paper presents the results of the estimation of the priming mixture force for primers used in 12.7 mm ammunition. The method of estimation is based on the results of pressure measurements in a closed chamber into which the products of the combustion of the priming mixture flowed. The capacity of the chamber was changed by inserting sleeves of various volumes. Another estimation of the force value was performed using the results of earlier investigations in which the chamber was filled with glass balls. The determined values of the force were compared with the values estimated based on the accessible literature data. The obtained estimations of the force value are of the same order of magnitude as the force value of black powder. This justifies the use of black powder characteristics for the assessment of the thermodynamic properties of priming mixture combustion products in interior ballistics calculations. The time of action of the investigated primer was determined using the optical recording of the outflow of the priming mixture combustion products into an open space. To facilitate the interpretation of the results of the experiments, a theoretical model was used. [ABSTRACT FROM AUTHOR]

Microsporidia are obligate intracellular pathogens that are currently considered to be most directly aligned with fungi. These fungal-related microbes cause infections in every major group of animals, both vertebrate and invertebrate, and more recently, because of AIDS, they have been identified as significant opportunistic parasites in man. The Microsporidia are ubiquitous parasites in the animal kingdom but, until recently, they have maintained relative anonymity because of the specialized nature of pathology researchers. Diagnosis of microsporidia infection from stool examination is possible and has replaced biopsy as the initial diagnostic procedure in many laboratories. These staining techniques can be difficult, however, due to the small size of the spores. The specific identification of microsporidian species has classically depended on ultrastructural examination. With the cloning of the rRNA genes from the human pathogenic microsporidia it has been possible to apply polymerase chain reaction (PCR) techniques for the diagnosis of microsporidial infection at the species and genotype level. The absence of genetic techniques for manipulating microsporidia and their complicated diagnosis hampered research. This study should provide basic insights into the development of diagnostics and the pitfalls of molecular identification of these ubiquitous intracellular pathogens that can be integrated into studies aimed at treating or controlling microsporidiosis. [ABSTRACT FROM AUTHOR]

Red algae (Rhodophyta) are a heterogeneous group of marine algal species that have served as a source of high-value molecules, including antioxidants and scaffolds, for novel drug development. However, it is challenging to identify Rhodophytes through morphological features alone, and in most instances, that has been the prevailing approach to identification. Consequently, this study undertook the identification of red algae species in Kenton-on-Sea, South Africa, as a baseline for future research on red algae biodiversity and conservation. The identification was achieved by designing, analysing, and using a set of universal primers through DNA barcoding of the rbc L gene. The PCR products of the rbc L gene were sequenced, and 96% of the amplicons were successfully sequenced from this set and matched with sequences on BOLD, which led to these species being molecularly described. Amongst these species are medicinally essential species, such as Laurencia natalensis and Hypnea spinella , and potential cryptic species. This calls for further investigation into the biodiversity of the studied region. Meanwhile, the availability of these primers will ease the identification process of red algae species from other coastal regions.

Treatment with pan-genotypic direct-acting antivirals, targeting different viral proteins, is the best option for clearing hepatitis C virus (HCV) infection in chronically infected patients. However, the diversity of the HCV genome is a major obstacle for the development of antiviral drugs, vaccines, and genotyping assays. In this large-scale analysis, genome-wide diversity and selective pressure was mapped, focusing on positions important for treatment, drug resistance, and resistance testing. A dataset of 1415 full-genome sequences, including genotypes 1-6 from the Los Alamos database, was analyzed. In 44% of all full-genome positions, the consensus amino acid was different for at least one genotype. Focusing on positions sharing the same consensus amino acid in all genotypes revealed that only 15% was defined as pan-genotypic highly conserved (≥99% amino acid identity) and an additional 24% as pan-genotypic conserved (≥95%). Despite its large genetic diversity, across all genotypes, codon positions were rarely identified to be positively selected (0.23%-0.46%) and predominantly found to be under negative selective pressure, suggesting mainly neutral evolution. For NS3, NS5A, and NS5B, respectively, 40% (6/15), 33% (3/9), and 14% (2/14) of the resistance-related positions harbored as consensus the amino acid variant related to resistance, potentially impeding treatment. For example, the NS3 variant 80K, conferring resistance to simeprevir used for treatment of HCV1 infected patients, was present in 39.3% of the HCV1a strains and 0.25% of HCV1b strains. Both NS5A variants 28M and 30S, known to be associated with resistance to the pan-genotypic drug daclatasvir, were found in a significant proportion of HCV4 strains (10.7%). NS5B variant 556G, known to confer resistance to non-nucleoside inhibitor dasabuvir, was observed in 8.4% of the HCV1b strains. Given the large HCV genetic diversity, sequencing efforts for resistance testing purposes may need to be genotype-specific or geographically tailored. [ABSTRACT FROM AUTHOR]

Adequate species' identification is critical for the detection and monitoring of biological invasions. In this study, we proposed and assessed the efficiency of newly created primer sets for the genetic identification of non-indigenous species (NIS) of fishes in the Volga basin based on: (a) a "long" fragment of cytochrome c oxidase subunit one of the mitochondrial gene (COI) (0.7 kb), used in "classical" DNA barcoding; (b) a short 3'-fragment (0.3 kb) of COI, suitable for use in high-throughput sequencing systems (i.e., for dietary analysis); (c) fragment of 16S mitochondrial rRNA, including those designed to fill the library of reference sequences for work on the metabarcoding of communities and eDNA studies; (d) a fragment of 18S nuclear rRNA, including two hypervariable regions V1-V2, valuable for animal phylogeny. All four sets of primers demonstrated a high amplification efficiency and high specificity for freshwater fish. Also, we proposed the protocols for the cost-effective isolation of total DNA and purification of the PCR product without the use of commercial kits. We propose an algorithm to carry out extremely cheap studies on the assessment of biological diversity without expensive equipment. We also present original data on the genetic polymorphism of all mass NIS fish species in the Volga-Kama region. The high efficiency of DNA identification based on our primers is shown relative to the traditional monitoring of biological invasions. [ABSTRACT FROM AUTHOR]

The reinvestigation of tetrazene single crystalline material by means of X-ray methods resulted in a slightly different structure when compared to previously published data. Reaction conditions responsible for different crystalline modification formation were investigated. Newly described C form was found to be the primary reaction product and the combined action of temperature and the presence of water over time is required for the transition to the A form. Both forms were described by X-ray powder diffraction. Tetrazene was also subjected to infrared and Raman spectroscopy, which allowed differentiating between the forms. The molecule was isotopically labeled with 15N atoms at two different locations (ring and nitrogen sidechain) and employed in assigning vibrational modes to the resulting bands. Differences between sensitivities to mechanical stimuli of the two modifications were investigated and found industrially insignificant. In the same vein, the performance of either modification in primer composition and primer was identical. [ABSTRACT FROM AUTHOR]

Banana mild mosaic virus (BanMMV) (Betaflexiviridae, Quinvirinae, unassigned species) is a filamentous virus belonging to the Betaflexiviridae family. It infects Musa spp. with a very wide geographic distribution. The genome variability of plant viruses, including the members of the Betaflexiviridae family, makes their molecular detection by specific primers particularly challenging. During routine indexing of the Musa germplasm accessions, a discrepancy was observed between electron microscopy and immunocapture (IC) reverse transcription (RT) polymerase chain reaction (PCR) test results for one asymptomatic accession. Filamentous viral particles were observed while molecular tests failed to amplify any fragment. The accession underwent high-throughput sequencing and two complete genomes of BanMMV with 75.3% of identity were assembled. Based on these sequences and on the 54 coat protein sequences available from GenBank, a new forward primer, named BanMMV CP9, compatible with Poty1, an oligodT reverse primer already used in diagnostics, was designed. A retrospective analysis of 110 different germplasm accessions from diverse origins was conducted, comparing BanMMCP2 and BanMMV CP9 primers. Of these 110 accessions, 16 tested positive with both BanMMCP2 and BanMMV CP9, 3 were positive with only BanMMCP2 and 2 tested positive with only BanMMV CP9. Otherwise, 89 were negative with the two primers and free of flexuous virions. Sanger sequencing was performed from purified PCR products in order to confirm the amplification of the BanMMV sequence for the five accessions with contrasting results. It is highly recommended to use the two primers successively to improve the inclusiveness of the protocol. [ABSTRACT FROM AUTHOR]

In the last decades, the evolution of molecular diagnosis methods has generated different advanced tools, like loop-mediated isothermal amplification (LAMP). Currently, it is a well-established technique, applied in different fields, such as the medicine, agriculture, and food industries, owing to its simplicity, specificity, rapidity, and low-cost efforts. LAMP is a nucleic acid amplification under isothermal conditions, which is highly compatible with point-of-care (POC) analysis and has the potential to improve the diagnosis in plant protection. The great advantages of LAMP have led to several upgrades in order to implement the technique. In this review, the authors provide an overview reporting in detail the different LAMP steps, focusing on designing and main characteristics of the primer set, different methods of result visualization, evolution and different application fields, reporting in detail LAMP application in plant virology, and the main advantages of the use of this technique. [ABSTRACT FROM AUTHOR]

Primers are used to reliably initiate a secondary explosive in a wide range of industrial and defence applications. However, established primer technologies pose both direct and indirect risks to health and safety. This review analyses a new generation of primer materials and ignition control mechanisms that have been developed to address these risks in firearms. Electrically or optically initiated metal, oxide and semiconductor-based devices show promise as alternatives for heavy metal percussive primers. The prospects for wider use of low-cost, safe, reliable and non-toxic primers are discussed in view of these developments. [ABSTRACT FROM AUTHOR]

The European Union (EU) is facing the recent arrival of the bacterium Xylella fastidiosa. Its fast spread has caused great alarm because of the economic impact it implies for the agroeconomy of European countries. Among its insect vectors, Philaenus spumarius has been demonstrated to transmit the bacterium from infected to uninfected trees in the EU, where different measures have been established to control it. One of the proposals to manage this vector is the augmentation of natural enemy populations. However, the identification of candidate predator species is essential if such a management system is to be introduced. The present paper describes a set of species-specific primers designed to detect the presence of P. spumarius DNA in soil arthropod fauna generalist predators’ gut which can reveal candidate species for the pest’s biological control. Such primers have been proven to be a useful and reliable taxonomic tool for P. spumarius identification at any life stage, i.e., nymphs. This rapid and accurate identification is essential for control strategies designed to avoid the spread of the pest and consequently the considerable economic losses it causes in crops. [ABSTRACT FROM AUTHOR]