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Abstract Comprehensive and accurate assessments of fish composition and diversity are essential for understanding fish ecology and resource management. Traditional fish surveys generally involve capturing organisms, which is invasive for the biological community under study and conflicts with the original intention of biodiversity conservation. Environmental DNA (eDNA) metabarcoding has become an integrated method for monitoring fish species without disturbing ecosystems. However, due to the serious nonspecific amplification of primers in eDNA‐based monitoring, many non‐fish sequences, usually human sequences, are also amplified, which results in serious data wastage and an increase in monitoring costs. We designed new universal primers for freshwater fish by analyzing the whole mitochondrial genome of Chinese freshwater fish. The performance of the primers was compared using an in silico polymerase chain reaction, followed by an in vitro metabarcoding analysis using eDNA from the Yangtze River, which is the third largest river in the world and harbors many freshwater fish species. We found that the mitochondrial 12S region is the most suitable metabarcoding gene marker for both Chinese and other freshwater fish. The minor change at the 3′‐end of the primer can greatly reduce the nonspecific amplification and improve the effectiveness of eDNA metabarcoding. Even small changes in primers may have qualitative and quantitative effects on the detected biodiversity, which should be considered in experimental design and data interpretation. These results will help with primer design and selection for eDNA‐based fish surveys, and consequently support the conservation of freshwater biodiversity.

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.

The article outlines the main DNA-based genetic molecular markers currently used to identify various plants, including fruit plants. It was noted that the choice of one or another marker depends on the execution technique and the characterized part of the genome. It also provides a brief description of DNA markers based on RFLP hybridization (RFLP), DNA markers based on PCR with primers that have multiple localization in the genome (multilocus markers) RAPD markers, AFLP - a method for obtaining molecular markers, DNA markers of unique SCAR sequences -markers, the principle of the CAPS technique, an important feature of micro-satellites based on SSR markers is noted. [ABSTRACT FROM AUTHOR]

The study, conducted at the research area of Raja Wala farm, University of Agriculture, Faisalabad, Pakistan, assessed the sunflowers' (Helianthus annuus L.) early maturity and yield improvement. Experimental material came from the United States Department of Agriculture and the National Agricultural Research Centre. Cytoplasmic male sterile lines and restorers, grown in the field, had their data gathered regarding early maturity. Then, the crossing of selected lines employed the line × tester design. The following season, the resulting crosses and their parents' evaluation proceeded in a randomized complete block design (RCBD) using three replications. The crosses declared as best hybrids in terms of early maturity and yield were 7-A × 86-R, 11-A × 83-R, 23-A × 81-R, 25-A × 80-R, 25-A × 94-R, and 27-A × 80-R. These best hybrids further underwent oil content and quality analysis. The crosses 23-A × 81-R and 25-A × 80-R revealed good performance for oil contents (palmitic, stearic, linoleic, and oleic acids) and quality traits like early maturing with better yield. Using RAPD markers, the authenticity assessment of the best hybrids through the presence and absence of bands compared with parents ensued. These hybrids will be helpful in future breeding programs for the development of early maturing varieties with improved achene yield and quality, which is rare in Pakistan. This material will also help develop the required hybrids. [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.

Soybean is an important agricultural product that has become widespread and popular due to its high nutritional qualities, namely protein content. One of the problems faced by commodity producers is a large number of so–called anti-nutritional proteins, trypsin and chymotrypsin inhibitors, due to the high concentration of which additional heat treatment is required before eating or for animal feed. Useful proteins include, in some cases, up to 70% of the total protein content of glycinins and b-conglycinins, which are a group of storage proteins. The creation of soybean varieties with an increased content of storing proteins requires a genetic analysis of the breeding material to identify the presence and level of expression of glycinins and b-conglycinins mRNA. This requires modeling of the most specific and not prone to the formation of secondary structures of primers. To model them, transcripts of the loci of interest for glycinins (Gy1-Gy5, Gy7) and β-conglycinins (CG-1, CG-4, CG-3) were found in the PlantsEnsemble database. The next step on the Unipro Ugene platform using the Primer3 toolkit was to select many pairs of primers for each of the loci, after which the pairs of primers were tested for specificity using Primer Blast from NCBI and for the formation of secondary structures (hairpins, homo- and heterodimers). As a result of the work done, pairs of primers were obtained that can be used to detect and determine the expression level of the genes of interest to us.

Purpose: To evaluate the influence of different ceramic surface conditioning methods on the fatigue failure load of adhesively cemented simplified lithium-disilicate glass-ceramic restorations. Materials and Methods: Ceramic (IPS e.max CAD, Ivoclar Vivadent) (Ø = 10 mm; thickness = 1.2 mm) and epoxy resin (Ø = 10 mm; thickness = 2.3 mm) disks were produced. The ceramic bonding surfaces were treated as follows: no etching and MPS-silane primer application only (MN); etching with 10% hydrofluoric acid (HF) for 20 s followed by primer application (HF + MN); HF + universal multimode adhesive application (HF + SU); etching with a one-step etching primer (ME&P); HF + primer + conventional adhesive (HF + MN + PAB). The epoxy resin disks were etched with 10% HF for 20 s followed by a coat of bonding agent (Multilink Primer A+B). Pairs of ceramic/epoxy resin disks were cemented with composite cement (Multilink N, Ivoclar Vivadent). The mean fatigue failure load was determined by the staircase method (100,000 cycles at 20 Hz frequency; initial load = 1435 N; step size = 72 N). Results: ME&P had the highest fatigue failure load, followed by HF etched groups, while the non-etched condition (MN group) had the lowest. All samples presented radial cracks originating from defects at the conditioned ceramic surface (interface).Conclusion: The simultaneous physicochemical conditioning with one-step self-etching ceramic primer promoted the best fatigue behavior results of the glass-ceramic restorations. It might indicate that this one-step conditioning reduces the number of flaws at the ceramic surface due to the slighter surface alterations than those produced by hydrofluoric acid etching, improving the fatigue behavior. [ABSTRACT FROM AUTHOR]

Montipora vietnamensis Veron, 2000 (Cnidaria, Anthozoa, Scleractinia, Acroporidae) is an uncommon, but distinctive species of stony coral. The complete mitochondrial genome of M. vietnamensis was sequenced in this study for the first time, based on 32 pairs of primers newly designed according to seven species in the family Acroporidae. The mitogenome of M. vietnamensis has a circular form and is 17,885 bp long, including 13 protein-coding genes (PCGs), 2 tRNA (tRNAMet, tRNATrp), 2 rRNA genes and a putative control-region. The base composition of the complete mitogenome was 24.8% A, 14.2% C, 24.2% G and 36.8% T, with a higher AT content (61.6%) than GC content (38.4%). Based on 13 protein-coding genes, a Maximum Likelihood phylogenetic analysis showed that M. vietnamensis is clustered in the genus Montipora which belongs to the family Acroporidae. More stony coral species should be sequenced for basic molecular information and to help confirm the taxonomic status and evolutionary relationships of Scleractinia in the future.

High-Risk Human Papillomavirus (HR-HPV) types 16 and 18 are estimated to be responsible for 72.4% of all HPV-related cancers worldwide in both men and women, including cervical, anal, penile, vulval, vaginal and head and neck cancers [1]. Important efforts worldwide have devoted to the study of these genotypes, throughout epidemiology and basic science approaches. Of particular interest are the genes from the early region (E), coding non-structural proteins. Early genes E1 and E2 products are involved in replication and transcription regulation, while E6 and E7 proteins are recognised for their oncogenic potential. In this data report, we described a set of primers based on reference sequences from HPV16 and HPV18 designed to cover the early region of these oncogenic genotypes. The design was based on multiple sequences alignment to identify the less conserved regions along the open reading frames (ORFs) E6, E7, E1 and E2. The design allows a highly stringent real time PCR essay ranged from 123 to 598 bp overlapping products for HPV16 (12 products in total) and from 183 to 526 bp for HPV18 (11 products in total), both spanning the early genomic region. The high annealing temperatures (Ta) and regions selected for primer bind were intended for genotypic specificity, without compromising the qPCR amplification efficiency (≥ 90%). Evaluation of qPCR conditions for primer set was performed using DNA standards as controls, generated from the HPV16 and 18 genomes clones. This provides relevant information for further multiple quantitative real-time PCR analysis (qPCR), using the SYBR green chemistry, which is is more affordable than generating multiple fluorescently labeled probes.

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]

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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]

Sealing bridge joints is important to prevent water, debris, and deicing corrosive materials to penetrate below the deck and damage the structure. A novel silicone foam sealant (SFS) has been developed to provide a long-term, cost-effective sealing method for small-movement expansion joints. This SFS exhibits a reduced elastic modulus which generates smaller stresses at the interface of the joint when the expansion joint widens in colder weather. This paper describes the experiments conducted to examine the bonding and ultimate stress–strain properties of the SFS compared to the commercially available product from which the SFS is derived. Factors studied include (a) substrate material, (b) presence of substrate primer, and (c) prolonged presence of saltwater. It was found that the SFS did not fail adhesively (i.e. no detachment from the substrate), compared to the commercial product which failed approximately 90% of the time via debonding at the interface to the substrate. On aging, the solid sealant exhibited significantly faster rate of (as much as three times) decrease in secant modulus over the 5-month saltwater aging period compared to the SFS. [ABSTRACT FROM AUTHOR]

The aim of the investigations is to reveal polymorphic microsatellite markers in order to study genetical diversity of sugar beet. Seedlings of sugar beet MS-lines and multi- and monogerm pollinators have been the material for the investigation. In experiments, 11 polymorphic Unigenes-markers and eight SSR-primers have been used to test starting materials. It has been determined that the length range of the obtained DNA-fragments is from 80 to 3000 bp. Most of the primers have provided stable amplification of DNA polymorphic fragments. The greatest level of polymorphism information content (PIC) has been determined for the loci identified using the following primers: Unigene 27 833 (PIC = 0.89), Unigene 2305 (PIC = 0.84), Unigene 17 623 (PIC = 0.84), Unigene 16 898 (PIC = 0.84), Unigene 24 552 (PIC = 0.64), Unigene 7492 (PIC = 0.82), FD1002 (PIC = 0.72), and Sb15 (PIC = 0.74). This enables clear differentiation of sugar beet breeding material. These loci have been related to various metabolic processes and play a large role in protective mechanisms of beet plants. The used primers make it possible to amplify up to 13 polymorphic bands per genotype. In Unigene 27 833, an SSR-locus, 13 PCR-products of 100–2800 bp in length have been identified. In total, 162 DNA-amplicons have been revealed. The value of polymorphism information content (PIC) is 0.89. It follows from the presented data that all nuclear microsatellite loci of the studied sugar beet samples included in the analysis show genetical variability, which allows for recommending them for use to identify genotypes of the crop. Based on the alleles revealed, the template of the investigated sugar beet samples' genetic affinity has been calculated, clusters have been constructed, and genetical distance (Euclidean) has been calculated. Genetical passports have been made according to the PAST algorithm. The greatest determined genetical distance (D) is 5.83 between an MS-form and the tetraploid pollinator developed by Lgovskaya Breeding Experimental Station. To produce sugar beet heterosis hybrids, parent pairs have been suggested taking into account distantly-related of initial forms. [ABSTRACT FROM AUTHOR]

DNA metabarcoding can be a promising alternative to microscopy for analysing phytoplankton, a key ecological indicator for freshwater ecosystems. The aim of this study was to evaluate the performance of different barcodes and associated primer pairs to assess microalgal diversity with DNA metabarcoding using a single barcode targeting all microalgae. We investigated barcodes in 16S and 23S rRNA genes, encoding for prokaryotic ribosomal sub-units, that are present in Cyanobacteria as well as in chloroplasts. In silico PCR tests were carried out on eight 16S and five 23S primer pairs using the Phytool reference library. Two and three pairs were selected for 16S and 23S, respectively, to perform an in vitro metabarcoding test based on a mock community made of DNA extracts of 10 microalgae strains. The 23S pairs enabled to detect all species, whereas 16S ones failed in the detection of some of them. One pair was selected for each genetic marker, based on its efficiency and specificity towards microalgae (e.g. not heterotrophic bacteria). Another mock community covering a larger diversity (18 microalgae strains) was used to test the efficiency of the selected pairs and their ability to estimate relative abundances. The 23S pair performed better than the 16S one for detecting target species with also more accuracy to assess their relative abundances. We conclude that the 23S primer pair ECLA23S_F1/ECLA23S_R1 appears as a good candidate to decipher freshwater phytoplankton communities. As a next step, it will be necessary to confirm these results on a large diversity of natural communities. Microalgae taxa can be identified with short DNA barcodes. Eight primer pairs for 16S and five for 23S were tested in silico and with mock communities including cyanobacteria and eukaryotic microalgae. We conclude that the 23S primer pair ECLA23S_F1/ECLA23S_R1 was the best candidate to decipher freshwater phytoplankton communities. [ABSTRACT FROM AUTHOR]

Acetic acid bacteria (AAB) are considered one of the most common wine spoilage microorganisms. They are still difficult to cultivate on laboratory media, which highlights the importance of alternative methods of detection of these bacteria. The goal of this work was development and testing of a fast and reliable Real Time Polymerase Chain Reaction (RT-PCR)-based method for easy detection of AAB in wine. We designed two primer sets for RT-PCR for detection of AAB and compared the results obtained using these primers with those obtained using a commercial kit. The results obtained using home designed primers showed good correlation with the results, obtained with the commercial screening kit.

This study aimed to design and evaluate a universal primer for Polymerase Chain Reaction (PCR)- based detection of mucormycosis-causing fungi by targeting their Internal Transcribed Spacer (ITS) sequences. In-silico analysis was conducted to assess primer suitability. Using Clustal Omega and Primer-BLAST, ITS sequences of 32 fungi species causing mucormycosis were aligned and subjected to primer design. Generated primers were sorted and in silico PCR simulations were performed to identify primers capable of amplifying all fungal species. Instead of identifying one pair of universal primer, in silico PCR analysis identified a panel of 14 primer pairs designed from full-length ITS sequences, and a panel of 12 primer pairs designed from conserved regions, that could detect 31 species. The study recommends a panel of 12 primers for multiplex-PCR to detect mucormycosis-causing fungi instead of a long list of PCR analyses for each fungus in diagnosing mucormycosis. [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.

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.

Simple sequence repeats of DNA (SSRs) are the most popular source of genetic markers used in population genetics, phylogenetics, and genetic mapping. A large number of nucleotide repeats enriched in G and C were identified. 336 mononucleotide motifs with more than ten repeats were recorded. 2020 nucleotide repeats were identified, of which 97.4% are di- (68.2%) and trinucleotides (29.2%). The total number of unique SSR loci, to which primers pairs were developed, was 1920. PCR primer sequences for unique SSR loci of the S. commune genome are presented. Of the twenty-two SSR markers synthesized for the S. commune genome, amplicons formed 64% on freshly isolated DNA samples.

Abstract Background This study aimed to investigate the genetic association of specific Single Nucleotide Polymorphisms (SNPs) within the muscle segment homeobox gene 1 (MSX1) with susceptibility to the peg-shaped teeth in 36 Jordanian Arab families and case–control samples in the Jordanian Arab population. Methods This cohort involved 108 individuals (36 trios families), which were used for family-based genetic study. Additionally, 56 patients and 57 controls were used for case–control study. Genomic DNA samples from both families and case–control were extracted according to distinguished processes. Then, polymerase chain reaction technique (PCR) was conducted using specific primers for the axons of the MSX1. Moreover, DNA sequencing genotyping method analysis of SNPs was used to detect specified SNPs in the MSX1 linked with peg-shaped teeth. Hardy–Weinberg Equilibrium and Chi-square were used to evaluate the data quality and the presence of any genotypic error. In addition, Transmission Disequilibrium Test (TDT) was used identify family-based association in which trios of parents and proband are used. Results The results of this study showed fourteen polymorphic sites in this gene, eight of them (rs121913129, rs104893852, rs104893853, rs121913130, rs104893850, rs1095, rs3775261, and rs1042484) were none-polymorphic. Meanwhile, the minor allele frequencies of the rest of the SNPs were polymorphic (rs8670, rs12532, rs3821949, rs4464513, rs1907998, and rs6446693). However, none of these SNPs were associated with peg-shaped teeth. Moreover, the haplotype genetic analysis revealed that there was no genetic association with peg-shaped teeth disorder susceptibility (P > 0.05) in the Jordanian families of Arab descent. Conclusions The present findings can be used in estimation of prevalence of peg-shaped teeth in the Jordanian population. However, our findings revealed that there is no evidence that the MSX1 polymorphisms had a crucial role in the peg-shaped teeth phenomenon, emphasizing that other genes might have this role. These findings are beneficial for clinicians to comprehensively understand the molecular aspects of teeth abnormalities.

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]

We aimed to evaluate whether two commonly used PCR primers are effective to identify P. endodontalis and discriminate from other prevalent black-pigmented bacteria in apical periodontitis (AP). Endodontic canal samples from patients with asymptomatic AP (n = 20) were collected and cultured in anaerobiosis. Two primer sets to detect P. endodontalis were selected from the literature and first analyzed for their specificity in silico; and then tested on clinical isolates in vitro and finally, in apical exudates ex vivo. The identity of P. endodontalis was verified by PCR and Sanger sequencing with universal primers for bacterial V3–V6 regions 16S rDNA. Only one primer set showed specificity only for P. endodontalis clones in silico and also was specific for P. endodontalis in vitro and ex vivo. [ABSTRACT FROM AUTHOR]

Palm trees are the third most important botanical family for humans because of their potential use in oils, drugs, cosmetics, food, and feed. Despite their importance, little information on their genetics and molecular variations exists, and a better understanding could contribute to breeding programs. This study aimed to determine the amount, distribution, and organization of plastid simple sequence repeats (cpSSRs) and their potential use in breeding in 52 species belonging to the order Arecales. Plastid genomes were analyzed to identify cpSSRs according to their nature, position, and presence in genic or intergenic regions. Primer pairs were validated in silico for amplification and polymorphisms in these SSRs and their dissimilarities were evaluated. The results showed a high frequency of mononucleotide repeats in the intergenic regions. Approximately 76 primer pairs were generated and are suggested for further studies. The dissimilarity analysis of cpSSRs showed that mono- and trinucleotides were highly abundant in plastid SSRs. [ABSTRACT FROM AUTHOR]

Allergens of food resources possess great variability at the proteomic and genomic sequences and an actual part of research activities because of the raising number of food allergies worldwide. Actually, only a few in silico based allergen nucleotide sequences comparing study exist, in spite of the great variability in the individual allergen families and with the regard to understand biological background of naturally hypoand hyperallergenic plants. The aim of the study was to analyze the mutation potential of allergen sequences variability relevant for primer design allergens in different plant species in comparison to the amaranthus, one of the allergenic safe specie, that provide many nutrition benefits and is an alternative for cereals. Here, NCBI GenBank Nucleotide and WHO/IUIS Allergens Nomeclature Sub-Commitee databases data mining were acquired a nucleotide sequence records of pollen allergens as bioinformatic data for evaluation of the potential of Amarathus spp. allergen. Data mining was performed in the nucleotide sequence records of cross-reactive pollen allergens (oleosins and profilins) created automatically with a computational algorithm translated from protein sequence records as bioinformatic material. 7 sequences of oleosins and 7 sequences of profilins were manually modified and analysed. A universal 3-step method of data processing identified and quantified mutations with a help of terms hierarchy. Definitively, the least substituted base is G, the most frequent substitution is T > C, and the most reproducible substitutions are C > G and G > C (profilins) and A > T (oleosins). The final description of sequence relationship is based on oleosin and profilin substitutions of 4 different glances (substitutions Profiles, single substitutions, group substitutions and fused substitutions) seeming Sal k 4 > Ole e 2 > Bet av 2 > Ama v 1 > Vac f 1 > Cro s 1 > Che a 1 > Koc s 2 > Pro j 1 > Ama r 1, where Sequence Pro j 1 is the most related to the matrix sequence of Ama r 1. The rising amount of sequence records in GenBank database will influence a sequential likelihood of conclusions at the time, but substitution frequency, sequence order, and reproducibility should be kept stable. [ABSTRACT FROM AUTHOR]

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., Competing Interests: The authors declare no conflict of interest.

An important approach of therapy the target gene sequence causes diseases via repair/recombine the mutated gene (gene transfer) using a restriction enzymes in the laboratory. This approach will cause multiple problems happening accompany to biological laboratory if ruled out problems outside of it like the digested DNA ran as a smear on an agarose gel, incomplete restriction enzyme digestion, extra bands in the gel, etc. The paper suggested new approach of therapy via repair/replacement mutated gene caused disease by detecting primers and finding restriction enzymes using bioinformatics tools, software, packages etc. then achieving the repair/ recombine of mutations before going to the biologic lab (out-lab) to avoid the problems associated these laboratories. Implement and apply this a proposed therapy approach on TP53 gene (which caused more than 50% of human cancers) and after confirming there is mutations on P53 tumor protein shows an effective cost, friendly therapy methodology and comprehensive.

• Micropropagation of micropropagation of Mansonia altissima (A. Chev.) A. Chev. var. altissima. • DNA Extraction of Wild plants and Direct micropropagation were performed. • PCR amplification and Gel electrophoresis were carried out. • Evaluation of somaclonal variation occurrence was assessed. Plant tissue culture that offers limited space and short time for clonal propagation of plant species can sometimes be discouraging if true to type clones are not generated. This study investigated direct shoot induction and genetic stability of Mansonia altissimia using molecular markers in order to ascertain that the in vitro propagated plantlets were true to type. Nodal cutting was used as explant, and Murashige and Skoog (MS) medium was supplemented with 6-benzyl aminopurine (BAP) (µM) + naphthaleneacetic acid (NAA) (µM), BAP (µM) + Gibberellic acid (GA 3) (µM), Kinetin (KIN) (µM) + NAA (µM), KIN (µM) + GA 3 (µM), thidiazuron (TDZ) (µM) + NAA (µM), TDZ (µM) + GA 3 (µM), for direct micropropagation. PCR amplification of wild plant and direct micropropagation of Mansonia altissimia were done using 15 RAPD and 6 ISSR primers. The highest shoot height (4.77 cm) and number of leaves (5.33) were obtained from 2.5 µM BAP + 3.0 µM GA 3. Moreover, the polymerase chain reaction amplification results showed 100% monomorphic and 1.0 level of similarity coefficients, which indicates that there is no occurrence of somaclonal variation. [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]

In recent decades publication of primers in local dialects has flourished in Estonia. The reasons for this process are related to the availability of financial support shemes for advancing regional culture as well as to identity creation on a more specific (local) level than just national identity. There are nine dialect primers issued in Estonia in 1998-2021. The initial inspiration for compiling such books comes from the ethnofuturist movement, but the reasons for publishing efforts are multiple, mostly related to the felt need of promoting local identity. The article describes the primers in a chronological order and proceeds to analyse them with the help of some concepts derived from semiotic theory. First, primers are regarded as intersemiotic phenomena combining verbal and pictorial information. Second, the concept of the chronotope of idyll, elaborated in the works of Mikhail Bakhtin, is applied in order to highlight the common features of the dialect primers, which are as follows: unity of place and time; cyclical pace of life; closeness to nature and the importance of natural sites and objects in the creation of human identity. Third, primers are related to Juri Lotman's ideas about two general types of culture, based respectively on oral and written transmission of information. Situated at an intersection of oral culture (dialect) and written culture (primers) these books pose complex challenges for analysis. Contentwise, dialect primers are based on the assumption that the socalled ethnographic time, featuring the lifestyle practised approximately at the end of the 19th century, is a particularly suitable frame for introducing local dialects to contemporary schoolchildren. In practice, 'gaps of time' occur in the texts as well as in illustrations that reveal the palimpsest-like texture of the chronotope of idyll featured in dialect primers. In conclusion it can be said that in addition to the effort of advancing pupils' linguistic abilities, it is the strengthening of local identity and intimate connection with the local natural environment that forms the basis of the dialect primers. [ABSTRACT FROM AUTHOR]

Abstract There is growing interest in the study of avian endoparasite communities, and metabarcoding is a promising approach to complement more conventional or targeted methods. In the case of eukaryotic endoparasites, phylogenetic diversity is extreme, with parasites from 4 kingdoms and 11 phyla documented in birds. We addressed this challenge by comparing different primer sets across 16 samples from 5 bird species. Samples consisted of blood, feces, and controlled mixes with known proportions of bird and nematode DNA. Illumina sequencing revealed that a 28S primer set used in combination with a custom blocking primer allowed detection of various plasmodiid parasites and filarioid nematodes in the blood, coccidia in the feces, as well as two potentially pathogenic fungal groups. When tested on the controlled DNA mixes, these primers also increased the proportion of nematode DNA by over an order of magnitude. An 18S primer set, originally designed to exclude metazoan sequences, was the most effective at reducing the relative number of avian DNA sequences and was the only one to detect Trypanosoma in the blood. Expectedly, however, it did not allow nematode detection and also failed to detect avian malaria parasites. This study shows that a 28S set including a blocking primer allows detection of several major and very diverse bird parasite clades, while reliable amplification of all major parasite groups may require a combination of markers. It helps clarify options for bird parasite metabarcoding, according to priorities in terms of the endoparasite clades and the ecological questions researchers wish to focus on.

Cloud computing technology got relevance as computing paradigm, which delivers massively scalable computing technologies based on the Internet for the organizations. Mobile Cloud Computing (MCC) allows users more possibilities to access services conveniently. But data security and privacy issues still act as barriers for adoption to cloud technology when dealing with confidential data. Encryption techniques play a significant role in ensuring data security in the Cloud. Various data encryption techniques have been evolved till the date of which DNA Computing techniques got wider acceptance due to biological complexity and computational properties of DNA. These features can be exploited to solve data security issues in MCC. This paper highlights the security issues in the MCC and proposes biocomputing solution approaches to address data security. A methodology is proposed based on the principle concepts of polymerase chain reaction and primer generation for ensuring data confidentiality and integrity. The security analysis of the proposed cryptosystem is evaluated by theoretical analysis, complexity and probability analysis. Formal analysis of the proposed protocol is done using Scyther and all the modeled claims are validated and positive results are obtained.

DNA barcoding methods are potentially useful for the detection and identification of species difficult to identify using morphology-based taxonomy, such as the larvae of marine taxa. Combining barcoding methods with massively parallel sequencing and low-cost DNA library preparation improves the feasibility of processing several samples but limits the taxonomic resolution of resulting sequences. In light of these limitations, we looked for suitable barcode regions for echinoderm larvae, first by examining the cytochrome c oxidase subunit I (COI) region, the most commonly used barcode for echinoderms. Using a dataset containing over 4000 COI sequences representing nearly 400 echinoderm species across all five extant classes, the COI gene was found to lack a sufficiently conserved region for barcoding. Taxonomic resolution was also found lacking, with no clear barcode gap across classes and the results of Automatic Barcode Gap Discovery (ABGD) failing to reflect current taxonomic assignments. While sequences may be mislabeled, the aggregation of multiple species in one ABGD partition suggests that course-grained taxonomic resolution may still result from using COI alone. The search for short alternative mitochondrial regions across 110 genomes of distinct species using ecoPrimers software revealed that class-specific primers targeting 12S and 16S ribosomal RNA (rRNA) regions are prime candidates for barcoding echinoderm sequences. The results of in silico PCR across the 110 mitochondrial genomes indicate that compared to existing COI primers, the candidate 12S and 16S primers yield similar taxonomic coverage, with slightly lower resolution. Taking into consideration the previous analysis of COI, we suggest the use of multiple markers (COI, 12S, 16S) to adequately capture the understudied diversity of echinoderm larvae. In vitro PCR of the 12S and 16S primers is also needed to validate the results of the in silico analyses [ABSTRACT FROM AUTHOR]

Oysters are major transmission vectors of noroviruses (NoVs) in the environment. Outbreaks of NoVs are often associated with the consumption of NoV-contaminated oysters. Laboratory confirmation of suspected oyster samples is a critical step in the surveillance and control of NoVs. Because of non-specific amplification, false-positive results are frequently obtained by semi-nested RT-PCR with the presently widely used primer set (G2SKF/G2SKR). Here, a novel universal PCR primer set N (NG2OF/NG2OR) specific for genogroup II (GII) NoVs was designed based on all GII NoV sequences available in public databases. Specific products were obtained with the primer set N when the NoV-positive oysters, spiked with each of five representative genotypes of GII NoVs (GII.17, GII.13, GII.4, GII.3, and GII.12), were subjected to analyzing. No products were detected with the primer set N for the NoV-negative oysters, while the primer set C gave various non-specific bands. Twenty-three out of 156 fresh oyster samples were NoV-positive with both the primer set N and the classic primer set, while eight were NoV-positive solely with the primer set N. Compared with the classic primer set, the newly designed primer set N had a higher detection rate and improved specificity for GII NoVs in oyster samples. These results show that the novel PCR primer pair is specific and applicable for the detection of GII NoVs in oysters. [ABSTRACT FROM AUTHOR]

Gene therapy is currently prohibited in human and equine athletes and novel analytical methods are needed for its detection. Most in vivo products use non‐integrating, recombinant viral vectors derived from adeno‐associated virus (AAV) to deliver transgenes into cells, where they are transcribed and translated into functional proteins. Although the majority of wild‐type AAV (WTAAV) DNA is removed from recombinant AAV (rAAV) vectors, some sequences are conserved. The goal of this study was to develop a quantitative polymerase chain reaction (QPCR) screening test targeting conserved AAV sequences to enable theoretical detection of all rAAV gene therapy products, regardless of encoded transgenes while excluding the presence of WTAAV DNA in horses. Primer sets were developed and validated to target an AAV2 sequence highly conserved across rAAV viral vectors and a sequence only found in wild type AAV2 (WTAAV2). Six horses were administered an intra‐articular injection of rAAV. Plasma and synovial fluid were collected on days 0, 1, 2, 4, 7, 14, 28, 56, and 84. Using QPCR, rAAV was detected in plasma for up to 2–4 days in all horses. rAAV DNA was detected for 28 days in synovial fluid from two horses for which synovial fluid samples were available. No WTAAV2 DNA was detected in any sample. This is the first study to develop a QPCR test capable of screening for rAAV vectors that may be used for gene doping in horses. [ABSTRACT FROM AUTHOR]

The article introduces some early Estonian primers recently discovered. So far the Estonian national bibliography was known to include five different primers - one of them survived in duplicate, the rest as single copies - printed before 1750, more specifically, from 1694 to 1741. Now, four new findings have been discovered, only one of which is similar to a copy known earlier. All four primers have survived as bound-with works in three bound-with volumes. Two of the volumes belonged to the library of the Earls of Macclesfield removed from Shirburn Castle, England, to be sold by Sotheby's in 2008. Through the auction, one of the volumes made its way to the US Congress Library and the other to the National Library of Poland. The former contains a primer published in the Tartu language (i.e. South Estonian) in 1724 and another printed in the Tallinn language (i.e. North Estonian) about 1700. The second copy of the latter is held at the Royal Library of Copenhagen. Both publications are reprints of primers compiled by Bengt Gottfried Forselius. The third volume with Estonian primers is located in the Gottfried Wilhelm Leibniz Library in Hannover. The covers hold two Latvian primers previously known and a Finnish primer published in Riga in 1694, unknown to the Finnish National Bibliography. The greatest surprise, however, were the undated Forselius primers printed in Riga, one presenting the Tallinn language and the other the Tartu language, both probably dating from before 1694. At least for the Tallinn version such dating is supported by orthographic analysis. The primers have probably been included in foreign collections as language examples. After all, the early modern period saw an increased research interest in the origin of peoples and languages, for which good reference material was provided by the cathechism texts of the primers. The Hannover primers could presumably be associated with the philological interests of Gottfried Wilhelm Leibniz, who was then in charge of the Hannover library. [ABSTRACT FROM AUTHOR]

Drought significantly reduces cowpea productivity. Information on genetic variation for differential expression of candidate genes for drought tolerance among cowpea genotypes, from which improvement plan could be drawn is limited in Nigeria. Variability of expression of the candidate gene NCED1 in cowpea was analyzed under different drought stress conditions. Primers based on NCED1 and P-Actin (used as an internal control) successfully amplified products from both stressed and unstressed accessions of cowpea. Contradictory responses were observed among drought-tolerant (mean STI > 0.57) and susceptible accessions (mean STI ˂ 0.57). NCED1 was significantly repressed by drought stress in all accessions, except in AC10, AC11, AC13 (tolerant accessions), and AC12 (susceptible accession). The results from stressed and unstressed conditions confirmed that the gene is expressed in both conditions. Biplot divided the accessions into four major groups, with most of the tolerant accessions in groups I and II, while most of the susceptible accessions occupied III and IV. Tolerant accessions such as AC22, AC15, AC23, AC13, AC10, AC11, and AC21 that combined higher plant height and dry root weight under drought stress with stress tolerance indices (STIs) possessed higher gene expression under both control and drought stress conditions. Therefore, positive correlations between the expression of the gene in both conditions and plant height under stress, on one hand, dry root weight under stress on the other hand, and the STIs confirm that its expression may be involved in drought tolerance of cowpea. Hence, the selection of cowpea based on higher levels of gene expression among accessions under both conditions may be effective for breeding drought-tolerant cowpea.

Aim: The aim of this study was to evaluate and compare the peel bond strength of an autopolymerizing acrylic resin and a fiberreinforced composite (FRC) resin to a heat temperature vulcanizing maxillofacial silicone (M511) using two different primers. Settings and Design: In vitro - comparative study. Materials and Methods: Autopolymerizing acrylic resin and FRC resin specimens with a dimension of 75 mm (length) ×10 mm (width) × 3 mm (height) were fabricated. A total of 60 samples were split into six categories based on the substructure material and primers (A330G primer and Sofreliner tough primer) used to bond the maxillofacial silicone to the FRC and acrylic resin specimens. In a universal testing machine, the peel bond strength was conducted at a 10 mm/min crosshead speed until bonding failure occurred. Statistical Analysis Used: The t-test, one-way analysis of variance, and the Tukey's honest significant difference (post hoc test) tests were used to statistically assess the values. Results: The Sofreliner tough primer produced the greatest peel bond strength in both the acrylic resin (0.89690 N/mm) and the FRC resin groups (3.19860 N/mm). Adhesive failures predominated in the acrylic resin group regardless of the primer used. The FRC group showed predominantly cohesive failures with both the A330G primer and Sofreliner tough primer. Conclusion: This study suggests that FRC resin combined with Sofreliner tough primer can significantly enhance the peel bond strength.

Designing primers for PCR-based taxonomic surveys that amplify a broad range of phylotypes in varied community samples is a difficult challenge, and the comparability of data sets amplified with varied primers requires attention. Here, we examined the performance of modified 16S rRNA gene and internal transcribed spacer (ITS) primers for archaea/bacteria and fungi, respectively, with nonaquatic samples. We moved primer bar codes to the 5' end, allowing for a range of different 3' primer pairings, such as the 515f/926r primer pair, which amplifies variable regions 4 and 5 of the 16S rRNA gene. We additionally demonstrated that modifications to the 515f/806r (variable region 4) 16S primer pair, which improves detection of Thaumarchaeota and clade SAR11 in marine samples, do not degrade performance on taxa already amplified effectively by the original primer set. Alterations to the fungal ITS primers did result in differential but overall improved performance compared to the original primers. In both cases, the improved primers should be widely adopted for amplicon studies. IMPORTANCE We continue to uncover a wealth of information connecting microbes in important ways to human and environmental ecology. As our scientific knowledge and technical abilities improve, the tools used for microbiome surveys can be modified to improve the accuracy of our techniques, ensuring that we can continue to identify groundbreaking connections between microbes and the ecosystems they populate, from ice caps to the human body. It is important to confirm that modifications to these tools do not cause new, detrimental biases that would inhibit the field rather than continue to move it forward. We therefore demonstrated that two recently modified primer pairs that target taxonomically discriminatory regions of bacterial and fungal genomic DNA do not introduce new biases when used on a variety of sample types, from soil to human skin. This confirms the utility of these primers for maintaining currently recommended microbiome research techniques as the state of the art.