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Background: Leptospirosis is a potentially life-threatening disease caused by bacteria of the genus Leptospira. The accurate identification and characterization of Leptospira species are critical for disease surveillance, outbreak investigation, and treatment strategies. Molecular techniques, such as Polymerase Chain Reaction (PCR) and Deoxyribonucleic acid (DNA) sequencing, have revolutionized the field of microbiology, providing rapid and accurate identification of Leptospira strains. However, optimizing these molecular techniques for accurate examination of Leptospira species can be challenging due to the genetic diversity and complexity of these bacteria. Purpose: This research aims to identify the most suitable primers for the precise identification of pathogenic Leptospira strains. Method: This research used the PCR method, using LipL32, rrs2, seqY, LipL41, IcdA, and Adk primers. A total of 17 isolates of pathogenic Leptospira bacteria were cultured from Institute of Vector Control and Reservoir Disease (IVRCD) in Salatiga, Indonesia. Result: The results of the research showed that the LipL41 and IcdA primers were found to be effective in distinguishing pathogenic strains, while the seqY, LipL32, Adk, and rrs2 primers required further refinement. The suitable Melting Temperature (TM) or annealing temperature is 58°C with 35 cycles of amplification. DNA concentration and purity had an A260/A280 ratio ranging between 1.8 and 2.8. Conclusion: LipL41 (500 bp) and IcdA (700 bp) are suitable primers for identifying pathogenic Leptospira.

Food is the primary cause for diseases in humans and carries high risk pathogens. Assessment of the safety in foods is needed to validate the presence of pathogenic bacteria. We used colony PCR for this approach to detect foodborne pathogens such as Escherichia coli, Lactobacillus and Bacillus cereus. Suitable primers were selected based on specific gene 1040 for Escherichia coli, gene S2 for Lactobacillus, and gene NVF for Bacillus cereus. Agarose gel electrophoresis is used for the detection of amplified products against a suitable marker. ImageJ is used for DNA band analysis, enabling precise quantification, normalization, and statistical comparisons. These studies have established a promising role in the detection of pathogens in various environmental samples. The insights gained from this study may serve as the foundation for rapid detection of foodborne diseases in the food industry.

Food is the primary cause for diseases in humans and carries high risk pathogens. Assessment of the safety in foods is needed to validate the presence of pathogenic bacteria. We used colony PCR for this approach to detect foodborne pathogens such as Escherichia coli, Lactobacillus and Bacillus cereus. Suitable primers were selected based on specific gene 1040 for Escherichia coli, gene S2 for Lactobacillus, and gene NVF for Bacillus cereus. Agarose gel electrophoresis is used for the detection of amplified products against a suitable marker. ImageJ is used for DNA band analysis, enabling precise quantification, normalization, and statistical comparisons. These studies have established a promising role in the detection of pathogens in various environmental samples. The insights gained from this study may serve as the foundation for rapid detection of foodborne diseases in the food industry. [ABSTRACT FROM AUTHOR]

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]

The test polypropylene substrates obtained from the original plastic car parts for Škoda Superb were subjected to 10 pre-treatment techniques: solvent cleaning (isopropanol, xylene), mechanical abrasion, immersion in a chromic acid mixture, flame activation, atmospheric and low-pressure plasma treatment, corona discharges and the application of two different primers (also called adhesion promoters). One of the primers, based on chlorinated polypropylene, was commercially available, while the other was prepared in our laboratory using polypropylene grafted with maleic anhydride. The test substrates were characterized using contact angle measurement for designation of surface free energy, Atomic Force Microscope (AFM) for roughness analysis, Scanning Electron Microscopy (SEM) for surface topography, and Energy Dispersive X-ray Spectrometry (EDX) for surface oxygen and contamination analysis. The suitability of the surface preparation methods in the industry was evaluated through peel strength tests (adhesive bonding process) and cross-cut tests (painting process). Not all tested methods were effective in increasing adhesion, especially in the adhesive bonding process, and even fewer in the painting process. However, some of them were suitable for both applications. The use of primers was found to be crucial. [ABSTRACT FROM AUTHOR]

Babaco is a hybrid cultivar native to the Andean region of Ecuador and Colombia, commercially attractive for its fruit. Babaco production in Ecuador faces losses from plant pathogens like babaco mosaic virus (BabMV), an RNA virus that causes chlorosis, leaf mottling, and deformation. Phylogenetic studies link BabMV to papaya mosaic virus (PapMV), alternanthera mosaic virus , and senna mosaic virus. To address this threat, we developed novel species-specific primers to detect BabMV targeting a 165 bp region of the coat protein (CP). Genus-specific primers were designed to validate the species-specific primers and attest their ability to discriminate between BabMV and its closest relatives. These primers targeted a 175 bp fragment of the CP region. The most effective sets of primers were chosen for reverse transcription polymerase chain reaction (RT-PCR) and SYBR® Green-based quantitative reverse transcription polymerase chain reaction (RT-qPCR) in symptomatic and asymptomatic babaco plants. Among 28 plants tested, 25 were positive and 3 were negative for BabMV using species-specific and genus-specific primers in RT-PCR and RT-qPCR, while the PapMV positive control was detected with the genus-specific primers and was negative for the species-specific primers. These primers represent a valuable molecular tool for detecting BabMV, potentially enhancing crop management. [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]

ABSTRACT Since the discovery of complete ammonia oxidizers (comammox) within the genus Nitrospira, their distribution and abundance across habitats have been intensively studied to better understand their ecological significance. Many primers targeting their ammonia monooxygenase subunit A gene (amoA) have been designed to detect and quantify comammox bacteria and to describe their community structure. We identified 38 published primers, but only few had high coverage and specificity for all known comammox Nitrospira or one of the two described subclades. For each target group, we comprehensively evaluated selected primer pairs using in silico analyses, endpoint PCRs, qPCRs, and amplicon sequencing on samples from various environments. Endpoint PCRs and qPCRs showed that the most commonly used primer pairs (comaA-244F/659R, comaB-244F/659R, and Ntsp-amoA162F/359R) produced several bands, which likely inflated quantifications via qPCR. In contrast, the recently published primer combinations CA377F/C576R, CB377F/C576R, and CA-CB377F/C576R resulted mostly in a single band. Furthermore, amplicon sequencing demonstrated that these primer combinations also captured the highest richness of comammox Nitrospira. Taken together, our results indicate that few existing comammox amoA primer combinations have both high specificity and coverage and that the choice of these high-specificity and high-coverage primer pairs substantially impacts the accurate detection, quantification, and community description of comammox bacteria. We, therefore, recommend using the CA377F/C576R, CB377F/C576R, and CA-CB377F/C576R primer pairs.IMPORTANCEBacteria that can fully convert ammonia via nitrite to nitrate, the complete ammonia oxidizers (comammox), were recently discovered and are found in many natural and engineered environments. PCR-based tools to study their abundance and diversity were rapidly developed, resulting in a plethora of primers available, many of which are widely used. The presence of comammox bacteria in an environment can, however, only be correctly determined if the used primers detect all members of this group while not detecting any other guilds. This study assesses the coverage and specificity of existing primers targeting comammox bacteria using both computational and standard molecular techniques, revealing large differences in their performance. The uniform usage of well-performing primers across studies could aid in generating comparable and generalizable data to better understand the importance of comammox bacteria in the environment.

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]

Molecular tools are an indispensable part of ecology and biodiversity sciences and implemented across all biomes. About a decade ago, the use and implementation of environmental DNA (eDNA) to detect biodiversity signals extracted from environmental samples opened new avenues of research. Initial eDNA research focused on understanding population dynamics of target species. Its scope thereafter broadened, uncovering previously unrecorded biodiversity via metabarcoding in both well‐studied and understudied ecosystems across all taxonomic groups. The application of eDNA rapidly became an established part of biodiversity research, and a research field by its own. Here, we revisit key expectations made in a land‐mark special issue on eDNA in Molecular Ecology in 2012 to frame the development in six key areas: (1) sample collection, (2) primer development, (3) biomonitoring, (4) quantification, (5) behaviour of DNA in the environment and (6) reference database development. We pinpoint the success of eDNA, yet also discuss shortfalls and expectations not met, highlighting areas of research priority and identify the unexpected developments. In parallel, our retrospective couples a screening of the peer‐reviewed literature with a survey of eDNA users including academics, end‐users and commercial providers, in which we address the priority areas to focus research efforts to advance the field of eDNA. With the rapid and ever‐increasing pace of new technical advances, the future of eDNA looks bright, yet successful applications and best practices must become more interdisciplinary to reach its full potential. Our retrospect gives the tools and expectations towards concretely moving the field forward. [ABSTRACT FROM AUTHOR]

Avian haemosporidian parasites are useful model organisms to study the ecology and evolution of parasite-host interactions due to their global distribution and extensive biodiversity. Detection of these parasites has evolved from microscopic examination to PCR-based methods, with the mitochondrial cytochrome b gene serving as barcoding region. However, standard PCR protocols used for screening and identification purposes have limitations in detecting mixed infections and generating phylogenetically informative data due to short amplicon lengths. To address these issues, we developed a novel genusspecific nested PCR protocol targeting avian haemosporidian parasites. The protocol underwent rigorous testing utilizing a large dataset comprising blood samples from Malagasy birds of three distinct Passeriformes families. Furthermore, validation was done by examining smaller datasets in two other laboratories employing divergent master mixes and different bird species. Comparative analyses were conducted between the outcomes of the novel PCR protocol and those obtained through the widely used standard nested PCR method. The novel protocol enables specific identification of Plasmodium, Haemoproteus (Parahaemoproteus), and Leucocytozoon parasites. The analyses demonstrated comparable sensitivity to the standard nested PCR with notable improvements in detecting mixed infections. In addition, phylogenetic resolution is improved by amplification of longer fragments, leading to a better understanding of the haemosporidian biodiversity and evolution. Overall, the novel protocol represents a valuable addition to avian haemosporidian detection methodologies, facilitating comprehensive studies on parasite ecology, epidemiology, and evolution. [ABSTRACT FROM AUTHOR]

Haploid-diploid life cycles impose unique eco-evolutionary consequences, rendering commonly used proxies difficult to use (e.g. separate sexes prevent selfing). Population genetic analyses are therefore required to explore patterns of reproductive system variation. However, there are still few haploiddiploid species for which polymorphic, nuclear loci exist. This problem is particularly acute for algae. Here, we describe the development of the first microsatellite loci in a freshwater red alga. We tested 73 candidate loci against a panel of Batrachospermum gelatinosum (L.) De Candolle gametophytes that encompass much of its North American range. Ten loci consistently amplified and were characterized by clean peak architectures on a capillary sequencer with one allele per locus, as expected in a haploid gametophyte. We then explored some basic population genetic indices in gametophytes collected from one site and obtained good resolution based on the probability of identity (pid). Yet, we observed a pattern of clumped repeated genotypes throughout the stream reach sampled. The pattern of moderate genotypic richness could be due to intragametophytic selfing resulting in the complete loss of genetic diversity from a single gamete union. Future studies will need to sample more populations to determine if intragametophytic selfing is the dominant reproductive mode in this monoicous taxon. The loci developed here represent an important tool for studying freshwater red algal populations in specific as well as enhancing our understanding of reproductive system variation and the haploid-diploid life cycle of algae in general. [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]

ABSTRACT Following the development of high‐throughput DNA sequencers, environmental prokaryotic communities were usually described by metabarcoding on short markers of the 16S domain. Among third‐generation sequencers, that offered the possibility to sequence the full 16S domain, the portable MinION from Oxford Nanopore was undervalued for metabarcoding because of its relatively higher error rate per read. Here we illustrate the limits and benefits of Nanopore sequencing devices by comparing the prokaryotic community structure in a mock community and 52 sediment samples from mangrove sites, inferred from full‐length 16S long‐reads (16S‐FL, ca. 1.5 kbp) on a MinION device, with those inferred from partial 16S short‐reads (16S‐V4V5, ca. 0.4 kbp, 16S‐V4V5) on Illumina MiSeq. 16S‐V4V5 and 16S‐FL retrieved all the bacterial species from the mock, but Nanopore long‐reads overestimated their diversity (56 species vs. 15). Whether these supplementary OTUs were artefactual or not, they only accounted for ca. 10% of the reads. From the sediment samples, with a coverage‐based rarefaction of reads and after singletons filtering, Mantel and Procrustean tests of co‐inertia showed that bacterial community structures inferred from 16S‐V4V5 and 16S‐FL were significantly similar, showing both a comparable contrast between sites and a coherent sea‐land orientation within sites. In our dataset, 84.7% and 98.8% of the 16S‐V4V5 assigned reads were assigned strictly to the same species and genus, respectively, than those detected by 16S‐FL. 16S‐FL detected 92.2% of the 309 families and 87.7% of the 448 genera that were detected by the short 16S‐V4V5. 16S‐FL recorded 973 additional species and 392 genus not detected by 16S‐V4V5 (31.5% and 10.4% of the 16S‐FL reads, respectively, among which 67.8% and 79.3% were assigned), produced by both primer specificities and different error rates. Thus, our results concluded an overall similarity between 16S‐V4V5 and 16S‐FL sequencing strategies for this type of environmental samples.

Grafting of vegetable crops is widely used in industrial vegetable growing in Asia and Western Europe. However, the influence of the genetic relationship of the scion and rootstock, the activity of peroxidase and the content of ascorbic acid in the components of the graft during inosculation period on graft survival has been poorly studied. The objects of the research are nine species of the pumpkin family (Cucurbitaceae). The polymorphism of 11 taxa was studied based on ISSR-PCR of genomic DNA. The record of survival rate of scion-rootstock combinations was conducted on the 10-th day. The activity of peroxidase was determined by the spectrophotometric method, the content of ascorbic acid was determined by Murri. Significant differences in survival rate were noted when grafting momordica into pumpkin rootstock variants: large-fruited (94.6 %) and nutmeg (67.1 %). In experiments with watermelon, melon and trichosanthes, the influence of the type of rootstock on the viability of the graft was not revealed. According to the ISSR spectra, the taxa were divided into two clusters; interspecific groupings of each clade were supported by average bootstrap values (from 26 to 100 %). A medium effect (r = 0.36) of the degree of genetic similarity of the scion with the rootstock on the survival rate of watermelon and a weak effect on the survival rate of melon, momordica and trichosanthes were revealed. The activity of peroxidase and the content of ascorbic acid in the grafting components in different scionrootstock combinations had a weak and moderate effect on plant survival, respectively. The survival rate of trichosanthes on various types of rootstocks strongly depended on the content of ascorbic acid (r = 0.7). Genetic relatedness, peroxidase activity, and ascorbic acid content had a weak or moderate effect on the survival rate of plants in scion-rootstock combinations.

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]

To evaluate the shear bond strengths and failure modes of dentin bonded to various restorative materials using three contemporary universal resin cements. Forty human third molars were longitudinally cut into 4 pieces. Ten sectioned were randomly divided into a group classified by bonding substrate materials and universal resin cement used. Two-mm diameter cylindrical rods were fabricated from five materials; Nickel-Chromium, Gold, Lithium disilicate glass ceramic, Zirconia, and Resin matrix ceramic. Three resin cements; Panavia V5, Duo-Link Universal, RelyX Universal were used to bond cylindrical specimens to prepared dentin surfaces according to the manufacturer's instructions. After subjected to thermocycling, shear bond strength was measured using a universal testing machine, and failure modes were assessed with a measuring microscope. Lithium disilicate glass ceramic displayed the highest bond strength of 36.14 MPa when bonded with Panavia V5, while Nickel-Chromium exhibited the highest bond strength of 38.02 MPa when bonded with Duo-Link Universal. The comparable bond strength could be obtained when using these two cements bonded Nickel-Chromium, Zirconia, and Lithium disilicate glass ceramic (p > 0.05). While, bonding to Gold and Resin matrix ceramic provided the inferior bond strength. No significant differences were found among all substrates when RelyX Universal cement was used (p > 0.05). The most common failure pattern was adhesive failure between restorative material and resin cement, followed by adhesive failure between dentin and resin cement. Gold and Resin matrix ceramic exhibited lower shear bond strength compared to the other materials, whereas Nickel-Chromium, Zirconia, and Lithium disilicate glass ceramic showed high shear bond strength when Panavia V5 and Duo-Link Universal were used. RelyX Universal showed lower shear bond strength than other cements and there were no significant differences among all substrates. [ABSTRACT FROM AUTHOR]

The major malaria mosquitoes, Anopheles gambiae s.l and Anopheles funestus, are some of the most studied organisms in medical research and also some of the most genetically diverse. When designing polymerase chain reaction (PCR) or hybridisation-based molecular assays, reliable primer and probe design is crucial. However, single nucleotide polymorphisms (SNPs) in primer binding sites can prevent primer binding, leading to null alleles, or bind suboptimally, leading to preferential amplification of specific alleles. Given the extreme genetic diversity of Anopheles mosquitoes, researchers need to consider this genetic variation when designing primers and probes to avoid amplification problems. In this note, we present a Python package, AnoPrimer, which exploits the Ag1000G and Af1000 datasets and allows users to rapidly design primers in An. gambiae or An. funestus, whilst summarising genetic variation in the primer binding sites and visualising the position of primer pairs. AnoPrimer allows the design of both genomic DNA and cDNA primers and hybridisation probes. By coupling this Python package with Google Colaboratory, AnoPrimer is an open and accessible platform for primer and probe design, hosted in the cloud for free. AnoPrimer is available here https://github.com/sanjaynagi/AnoPrimer and we hope it will be a useful resource for the community to design probe and primer sets that can be reliably deployed across the An. gambiae and funestus species ranges.

Avian haemosporidian parasites are useful model organisms to study the ecology and evolution of parasite-host interactions due to their global distribution and extensive biodiversity. Detection of these parasites has evolved from microscopic examination to PCR-based methods, with the mitochondrial cytochrome b gene serving as barcoding region. However, standard PCR protocols used for screening and identification purposes have limitations in detecting mixed infections and generating phylogenetically informative data due to short amplicon lengths. To address these issues, we developed a novel genus-specific nested PCR protocol targeting avian haemosporidian parasites. The protocol underwent rigorous testing utilizing a large dataset comprising blood samples from Malagasy birds of three distinct Passeriformes families. Furthermore, validation was done by examining smaller datasets in two other laboratories employing divergent master mixes and different bird species. Comparative analyses were conducted between the outcomes of the novel PCR protocol and those obtained through the widely used standard nested PCR method. The novel protocol enables specific identification of Plasmodium, Haemoproteus (Parahaemoproteus), and Leucocytozoon parasites. The analyses demonstrated comparable sensitivity to the standard nested PCR with notable improvements in detecting mixed infections. In addition, phylogenetic resolution is improved by amplification of longer fragments, leading to a better understanding of the haemosporidian biodiversity and evolution. Overall, the novel protocol represents a valuable addition to avian haemosporidian detection methodologies, facilitating comprehensive studies on parasite ecology, epidemiology, and evolution.

Microbes produce various types of volatile organic compounds (VOCs) through metabolism, which can be used for diagnostic purposes. Microbes' types and classes of VOCs are very wide, including fatty acid derivatives (hydrocarbons, alcohols, and ketones), aromatic compounds, nitrogen-containing compounds, and volatile sulfur compounds. Microbial volatile organic compounds (VOCs) can also be divided into several chemical classes: alkenes, alcohols, ketones, benzos, pyrazines, sulfides, acids, esters, and terpenes. This study aimed to design primers for genes encoding volatile synthesis in Ralstonia syzygii subsp. celebesensis, which causes blood disease in the banana plant. Some of the genes involved are adc (acetone synthesis), adhP (ethanol synthesis), ilvA, nirBD (ammonia synthesis), mdcA (propionic acid synthesis), cysI (hydrogen sulfide synthesis), and speBC (putrescine synthesis). Primers were designed and examined for specificity in silico using Primer3Plus, Geneious Prime, and BLAST programs. The numbers of nine pairs designed primers were successfully amplifying the related nine VOC genes of R. syzygii subsp. celebesensis for qPCR.

A wild elephant calf of 6-8 months of age was brought to the Centre for Wildlife Rehabilitation and Conservation (CWRC), Bokakhat, Assam, India, having symptoms of diarrhea and debility. The fecal sample was collected and studied for isolation and identification of bacteria. Eosin Methylene Blue (EMB) agar media showed moderate growth of bacterial colonies with a characteristic green metallic sheen. The isolates were subjected to Gram stain and biochemical tests (lactose, sucrose, and sorbitol). Polymerase chain reaction (PCR) amplification with universal primers showed an amplicon of 300 bp. The PCR amplified product after nucleotide BLAST showed 98.95 % similarity with the sequence of enteropathogenic Escherichia fergusonii.

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.

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.

Relevance. Hepatitis B virus (HBV) is one of the most common viral infections affecting people worldwide and can lead to chronic hepatitis, cirrhosis and hepatocellular carcinoma. Currently 3% of the world's population are infected with hepatitis B virus and are at risk of developing life-threatening liver disease. Immunological and molecular biological methods of detection of HBV are currently used in laboratory diagnostics. The polymerase chain reaction (PCR) is currently the most sensitive method for the detection and quantification of HBV. HBV DNA quantification is widely used to monitor the antiviral treatment of HBV infection.Aim. To develop a real-time PCR kit for the quantification of HBV DNA.Materials and methods. A total of 200 plasma and serum samples positive and negative for HBV were used in the development. The performance of the developed kit was compared with the use of other commercially registered HBV diagnostic kits in Russia. Additionally, the nucleotide sequences of all existing virus genotypes analysed for the selection of primers using GeneBank system.Results and discussion. Comparison analysis of the results of quantitative determination by real-time PCR in 200 clinical serum and blood plasma samples showed that the diagnostic sensitivity of the developed kit was 100% and specificity 100%. The primers developed specific to the POL gene region. The kit is capable of detecting all types of virus genotypes.Conclusions. The developed reagent kit allows detection of hepatitis B virus and determination of its quantity within 70 minutes. In addition to a large number of genotypes and subgenotypes, the virus is characterized by mutational changes in the genome, which complicates its diagnosis and, as a consequence, the ongoing therapy with drugs. Conservative regions for primer and probe selection taken into account in the development, and the sequencing results obtained are applicable to all HBV genotypes. The reagent kit is designed to monitor HBV infected patients and will allow the analysis of different HBV viral loads.

Ecosystem health can be monitored by analyzing the species diversity and abundance in a region. The process of recognizing species based on morphology takes a lot of time and expertise. Metabarcoding and other modern molecular taxonomic methods can help speed up species identification. However, the choice of gene markers and primers, the lack of reference sequences in public databases, and the choice of tools used in bioinformatic analyses have a substantial impact on the observed diversity. To test the effectiveness of metabarcoding in assessing the species diversity, mock communities of zooplankton species found in the Saudi Arabian Gulf were assembled and subjected to high-throughput sequencing (HTS) using an Illumina MiSeq platform. Short CO1 and 28S nuclear gene fragments, containing the D2 region, of lengths 313 bp and 400 bp, respectively, were used as gene markers. Trimmomatic, Cutadapt, the FLASH read-merge software tool, and the USEARCH pipeline for OTU clustering were used in the bioinformatic analyses. Around 90% of the zooplankton species in the mock communities were detected. We conclude that the combination of CO1 and 28S markers is a quick and effective tool for evaluating zooplankton species diversity.

Background and Aim: Equine influenza (EI) is a highly contagious disease that causes fever and upper respiratory tract inflammation. It is caused by influenza virus A, belonging to the Orthomyxoviridae family, with subtypes H3N8 and H7N7. This study presents data on the development of a real-time polymerase chain reaction (RT-PCR) assay using TaqMan probes to detect the H3 subtype of EI virus (EIV). Materials and Methods: The evaluation of the developed RT-PCR assay involved five strains of EIV as positive controls and ten nasopharyngeal swab samples collected from horses. RNA was isolated using the GeneJet Viral DNA and RNA Purification Kit, and primers and probes were designed using the Integrated DNA Technology PrimerQuest Tool. The assay was optimized by investigating the annealing temperature, primer and probes concentrations, sensitivity, and specificity. Sequencing was performed using the Thermo Fisher 3130 Genetic Analyzer, and the evolutionary history was inferred using the Neighbor-Joining method. Results: The designed primers and probes, targeting the H3 gene, were found to be specific to the EIV. The RT-PCR assay was capable of detecting as low as 50 femtogram (f) or 3 × 103 copies of genomic RNA. No cross-reactions were observed with other respiratory viral and bacterial pathogens, indicating the high specificity of the assay. To evaluate its effectiveness, ten nasopharyngeal swab samples collected from farms in North Kazakhstan regions during disease monitoring were analyzed. The accuracy of the analysis was confirmed by comparing the results with those obtained from a commercial RT-PCR assay for EI identification. The developed RT-PCR assay exhibited high sensitivity and specificity for detecting the EIV. Conclusion: The results demonstrate that the developed RT-PCR assay is suitable for diagnosing EI. This simple, highly sensitive, and specific assay for detecting H3 EIV can be a reliable tool for diagnosing and surveilling EI. Implementing this RT-PCR assay in veterinary practice will enhance and expedite the timely response to potential outbreaks of EI, thus positively impacting the overall epizootic well-being of EI in Kazakhstan.

La adhesión efectiva de las restauraciones de zirconio y otros metales a los cementos de resina es crucial en la odontología. La falta de una adhesión adecuada puede llevar al fracaso en el tratamiento de rehabilitación oral. El objetivo fue realizar una revisión bibliográfica sobre el impacto de los primers de metal en el zirconio y otros metales. El estudio corresponde con una revisión bibliográfica narrativa de la literatura de publicaciones ubicadas en las bases de datos de PubMed, Web of Science y Scopus; el universo fue de 89 estudios y una muestra final de 54 publicaciones relevantes según criterios de inclusión y exclusión. Entre los principales resultados se tiene que el uso de primers específicos, como el agente 10-MDP, mejora significativamente la fuerza de adhesión entre el zirconio y los cementos de resina. Además, se destaca la importancia de la preparación adecuada de la superficie del zirconio para lograr una adhesión óptima. Estos resultados respaldan la selección cuidadosa de primers y la implementación de técnicas de preparación adecuadas para garantizar una adhesión efectiva en las restauraciones dentales con zirconio. En conclusión, los hallazgos subrayan la importancia de utilizar primers específicos, como el agente 10-MDP, y llevar a cabo una preparación adecuada de la superficie del zirconio para lograr una adhesión óptima en restauraciones dentales.

The first textbook for Spanish learners of Chinese was published in Madrid in 1861. It epitomises how Euro-American linguists abandoned their focus on classical language and took a new approach towards practical modern Chinese. This article provides a reflection on why that linguistic transition took place, how language became a key battlefield in the imperial game in China and what the relevance of intellectual factors was in the colonial enterprise. Moreover, through the analysis of that textbook, it questions the relevance of nations, such as Spain, that played a peripheral role in the process of the imperial siege of China. These countries did not limit themselves to mimic the actions of great imperial powers but tried to find their own path in the Chinese colonial scenario, as the case of El intérprete chino illustrates. [ABSTRACT FROM AUTHOR]

A wild elephant calf of 6-8 months of age was brought to the Centre for Wildlife Rehabilitation and Conservation (CWRC), Bokakhat, Assam, India, having symptoms of diarrhea and debility. The fecal sample was collected and studied for isolation and identification of bacteria. Eosin Methylene Blue (EMB) agar media showed moderate growth of bacterial colonies with a characteristic green metallic sheen. The isolates were subjected to Gram stain and biochemical tests (lactose, sucrose, and sorbitol). Polymerase chain reaction (PCR) amplification with universal primers showed an amplicon of 300 bp. The PCR amplified product after nucleotide BLAST showed 98.95 % similarity with the sequence of enteropathogenic Escherichia fergusonii. [ABSTRACT FROM AUTHOR]

In the course of the study, specific primers were developed to identify 20 species of phytopathogenic bacteria from 12 genera associated with the occurrence of infectious diseases in seven forest-forming species of Belarus. The results showed that the developed oligonucleotides are specific for the 16S (mtDNA and cpDNA) and 23S rRNA genes of the studied phytopathogenic bacteria. At a further stage of research, in vitro tests will be carried out to establish the effectiveness of diagnosing bacterial marker loci, both using pure cultures of pathogen isolates and samples of affected tissues of woody plants. [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]

Colorectal cancer (CRC) is a leading cause of cancer death in the USA. Screening programs in federally qualified health centers (FQHCs) are essential to reducing CRC-related mortality and morbidity among underserved populations. Centralized, population-based mailed fecal immunochemical test (FIT) programs can improve CRC screening rates, but barriers to implementation remain. We qualitatively explored barriers and facilitators to implementation of a mailed FIT program at a large, urban FQHC that employed advance notification "primers" (live calls and texts) and automated reminders. We interviewed 25 patients and 45 FQHC staff by telephone about their experience with the program. Interviews were transcribed, coded, and content analyzed using NVivo.12. Patients and staff found advance notifications conveyed through live phone calls or text messages to be acceptable and motivational for FIT completion. Live phone primers were helpful in addressing patients' questions and misconceptions about screening, particularly for patients new to screening. Advance notifications sent by text were considered timely and useful in preparing patients for receipt of the FIT. Barriers to implementation included lack of receipt of primers, reminders, or the mailed FIT itself due to inaccurate patient contact information within the FQHC medical record; lack of systems for documenting mailed FIT outreach to coordinate with clinical care; and lack of local caller identification for primers and reminders. Our findings demonstrate that an enhanced mailed FIT program using primers and reminders was acceptable. Our findings can help other FQHCs implement and optimize their mailed FIT programs. [ABSTRACT FROM AUTHOR]

Molecular genetic markers associated with saltpan effluents (NaCl) on two genotypes Cajanus cajan and Sorghum bicolor crop plants were studied in four different field Areas at vegetative stage, anthesis phase and seed set phase of plant growth. Among the 10 primers used only 5 primers produced reproducible and consistent banding pattern, those are OPA-17, OPE-14, OPE-15, OPE-17 and OPA-16. In that OPE-14 produced height 24 bands. And OPA-17 produced 16 bands and in that 14 were polymorphic bands and the percentage of polymorphism was 87.5%. PCR success rate for rbcl was 100% and sequence success rate was also 100% success. BLAST results revealed that the three species sequence for rbcl loci was 98% similar. Finally, the present study revealed the existence of considerable variation at the molecular level in the Cajanus cajan germplasm rather than in germplasm of Sorghum bicolor, which confirms the results of the earlier on morphological and biochemical studies. [ABSTRACT FROM AUTHOR]

The expediency of using unique SSR DNA-markers of Schizophyllum commune for population genetic assays in various fungal species has been demonstrated. In Auricularia auricula-judae and Irpex lacteus, we observed formation of heterogeneous amplicons mostly up to 500 bp in length that ensured their high resolution and facilitated data analysis. The established sets of molecular markers are efficient for DNA-fingerprinting of S. commune, I. lacteus, and A. auricula-judae, as well as are prospective for species of the genus Pleurotus, but need to be further enlarged.

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.

Calanoid copepod populations are being severely affected due to the effects of ocean acidification (OA) and ocean warming (OW). These marine organisms are the most abundant primary consumers contributing significantly in the marine food web. Any effect on the abundance and diversity of copepods due to climate change is likely to have serious implications on the marine ecosystem functioning. Molecular studies that play a vital role in assessing the genetic changes under the influence of environmental imbalances are completely lacking for this species. Here we report the genetic variations in three generations of copepods through transcriptome sequencing. RNA sequencing was performed on an Illumina HiSeq platform employing the 2 × 100 bp paired-end chemistry. Approximately, 10GB of data was obtained for all the samples. The raw sequences were assembled through Trinity 2.6.6 and mined for single nucleotide polymorphisms (SNPs) and simple sequence repeats (SSRs). MIcroSAtellite Identification Tool (MISA) was used for SSR detection and Primer 3 (v 3.0) was utilized to design short oligonucleotide primers (18-20 mers). A total of 15,222 SSRs were identified and 28,944 primer pairs were designed against these motifs. The transcriptome possessed 413,890 SNPs at a frequency of 2.8 per kb. The newly discovered SSRs and SNPs could act as genetic markers for future studies on genetic diversity and conservation for Parvocalanus crassirostris.

Ecosystem health can be monitored by analyzing the species diversity and abundance in a region. The process of recognizing species based on morphology takes a lot of time and expertise. Metabarcoding and other modern molecular taxonomic methods can help speed up species identification. However, the choice of gene markers and primers, the lack of reference sequences in public databases, and the choice of tools used in bioinformatic analyses have a substantial impact on the observed diversity. To test the effectiveness of metabarcoding in assessing the species diversity, mock communities of zooplankton species found in the Saudi Arabian Gulf were assembled and subjected to high-throughput sequencing (HTS) using an Illumina MiSeq platform. Short CO1 and 28S nuclear gene fragments, containing the D2 region, of lengths 313 bp and 400 bp, respectively, were used as gene markers. Trimmomatic, Cutadapt, the FLASH read-merge software tool, and the USEARCH pipeline for OTU clustering were used in the bioinformatic analyses. Around 90% of the zooplankton species in the mock communities were detected. We conclude that the combination of CO1 and 28S markers is a quick and effective tool for evaluating zooplankton species diversity. [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]

Background and Aim: Equine influenza (EI) is a highly contagious disease that causes fever and upper respiratory tract inflammation. It is caused by influenza virus A, belonging to the Orthomyxoviridae family, with subtypes H3N8 and H7N7. This study presents data on the development of a real-time polymerase chain reaction (RT-PCR) assay using TaqMan probes to detect the H3 subtype of EI virus (EIV). Materials and Methods: The evaluation of the developed RT-PCR assay involved five strains of EIV as positive controls and ten nasopharyngeal swab samples collected from horses. RNA was isolated using the GeneJet Viral DNA and RNA Purification Kit, and primers and probes were designed using the Integrated DNA Technology PrimerQuest Tool. The assay was optimized by investigating the annealing temperature, primer and probes concentrations, sensitivity, and specificity. Sequencing was performed using the Thermo Fisher 3130 Genetic Analyzer, and the evolutionary history was inferred using the Neighbor-Joining method. Results: The designed primers and probes, targeting the H3 gene, were found to be specific to the EIV. The RT-PCR assay was capable of detecting as low as 50 femtogram (f) or 3 × 10³ copies of genomic RNA. No cross-reactions were observed with other respiratory viral and bacterial pathogens, indicating the high specificity of the assay. To evaluate its effectiveness, ten nasopharyngeal swab samples collected from farms in North Kazakhstan regions during disease monitoring were analyzed. The accuracy of the analysis was confirmed by comparing the results with those obtained from a commercial RT-PCR assay for EI identification. The developed RT-PCR assay exhibited high sensitivity and specificity for detecting the EIV. Conclusion: The results demonstrate that the developed RT-PCR assay is suitable for diagnosing EI. This simple, highly sensitive, and specific assay for detecting H3 EIV can be a reliable tool for diagnosing and surveilling EI. Implementing this RT-PCR assay in veterinary practice will enhance and expedite the timely response to potential outbreaks of EI, thus positively impacting the overall epizootic well-being of EI in Kazakhstan. [ABSTRACT FROM AUTHOR]

Microbes produce various types of volatile organic compounds (VOCs) through metabolism, which can be used for diagnostic purposes. Microbes' types and classes of VOCs are very wide, including fatty acid derivatives (hydrocarbons, alcohols, and ketones), aromatic compounds, nitrogen-containing compounds, and volatile sulfur compounds. Microbial volatile organic compounds (VOCs) can also be divided into several chemical classes: alkenes, alcohols, ketones, benzos, pyrazines, sulfides, acids, esters, and terpenes. This study aimed to design primers for genes encoding volatile synthesis in Ralstonia syzygii subsp. celebesensis, which causes blood disease in the banana plant. Some of the genes involved are adc (acetone synthesis), adhP (ethanol synthesis), ilvA, nirBD (ammonia synthesis), mdcA (propionic acid synthesis), cysI (hydrogen sulfide synthesis), and speBC (putrescine synthesis). Primers were designed and examined for specificity in silico using Primer3Plus, Geneious Prime, and BLAST programs. The numbers of nine pairs designed primers were successfully amplifying the related nine VOC genes of R. syzygii subsp. celebesensis for qPCR. [ABSTRACT FROM AUTHOR]

Background: Leishmania is the parasitic protozoan responsible for leishmaniases, a disease that can cause a range of cutaneous, mucosal, and visceral infections. Two subgenera L. Viannia and L. Leishmania are known to infect humans in the tropics and subtropics of the Americas. The aim of the present study was to develop a new pair of primers for the two subgenera and test in clinical samples. Methods: We designed two new pairs of primers for a PCR method from two conserved genes, cysteine proteinase B (cpb) and N-acetylglucosamine-6-phosfate deacetylase- like protein (nagA), as specific markers for those two respective subgenera. Primers were tested with 16 microscopical positive clinical samples from the Amazon region of Ecuador obtained in 2010-2020 period. Results: The cpb presented a band of 172 bp and the nagA a band of 300 bp, thus clearly differentiating L. viannia from L. leishmania. Additionally, primers identified and differentiated the clinical samples in the two subgenera. Conclusion: The new primers targeting different two genes and standardized in a PCR assay could identified and differentiated Leishmania parasites at subgenus level. This protocol could be used for Leishmania genus identification and diagnosis at the subgenus level and for determining the parasite's geographical distribution where different Leishmania subgenera are found in the same area. [ABSTRACT FROM AUTHOR]