Journal articles: 'Primers'

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Semenov, V. M., S. K. Yahorau, I. A. Lyatos, T. I. Dmitrachenko, A. A. Marchenko, M. S. Kosova, S. K. Zenkova, and K. A. Savochkina. "REAL-TIME PCR TEST SYSTEM FOR TTV DNA DETECTION IN BIOLOGICAL MATERIAL." Hepatology and Gastroenterology 8, no. 1 (June 10, 2024): 36–41. http://dx.doi.org/10.25298/2616-5546-2024-8-1-36-41.

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Background. The study of biological material for the presence of TTV DNA using the PCR method allows for a timely assessment of the functional state of the human liver and immune system. Objective. To develop components for real-time PCR for TTV DNA detection in biological material. Material and methods. The design and selection of optimal primers and probes (taking into account the size (length) of the amplicon, annealing temperature, nucleotide composition, distribution of nucleotides along the length of the primer, length of primers, the possibility of formation of hairpins and dimers by primers) were performed using the Primer-BLAST/Primer3, FastPCR programs. Since primers, even absolutely unique for certain DNA sequences, could anneal at nonspecific sites, not related to the gene analyzed, we checked the correspondence of the primers to the sequences of the target gene. For this purpose, we used the NCBI Primer BLAST online service and assessed the local pairwise alignment of each primer with all nucleotide sequences of the Refseq databases. Results. As the result of studies carried out on the selection of the optimal primer annealing temperature, primer concentrations, as well as the selection of the optimal nucleotide pair, the main parameters of the designed primers were determined. Conclusions. A kit for the detection and quantification of TTV DNA using the polymerase chain reaction method with hybridization-fluorescent detection in real time was created and became the basis for the development of a commercial test system.

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Levi, Amnon, William P. Wechter, Karen R. Harris, Angela R. Davis, and Zhangjun Fei. "High-frequency Oligonucleotides in Watermelon Expressed Sequenced Tag-unigenes Are Useful in Producing Polymorphic Polymerase Chain Reaction Markers among Watermelon Genotypes." Journal of the American Society for Horticultural Science 135, no. 4 (July 2010): 369–78. http://dx.doi.org/10.21273/jashs.135.4.369.

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In this study, we report a simple procedure for developing and using new types of polymerase chain reaction (PCR) primers, named “high-frequency oligonucleotides–targeting active genes” (HFO-TAG). The HFO-TAG primers were constructed by first using a “practical extraction and report language” script to identify oligonucleotides (8, 9, and 10 bases) that exist in high frequency in 4700 expressed sequence tag (EST)-unigenes of watermelon (Citrullus lanatus) fruit. This computer-based screening yielded 3162 oligonucleotides that exist 32 to 335 times in the 4700 EST-unigenes. Of these, 192 HFO-TAG primers (found 51 to 269 times in the 4700 EST-unigenes) were used to amplify genomic DNA of four closely related watermelon cultivars (Allsweet, Crimson Sweet, Charleston Gray, and Dixielee). The average number of DNA fragments produced by a single HFO-TAG primer among these four watermelon cultivars was considerably higher (an average of 5.74 bands per primer) than the number of fragments produced by intersimple sequence repeat (ISSR) or randomly amplified polymorphic DNA (RAPD) primers (an average of 2.32 or 4.15 bands per primer, respectively). The HFO-TAG primers produced a higher number of polymorphic fragments (an average of 1.77 polymorphic fragments per primer) compared with the ISSR and RAPD primers (an average of 0.89 and 0.47 polymorphic fragments per primer, respectively). Amplification of genomic DNA from 12 watermelon cultivars and two U.S. Plant Introductions with the HFO-TAG primers produced a significantly higher number of fragments than RAPD primers. Also, in PCR experiments examining the ability of primers to amplify fragments from a watermelon cDNA library, the HFO-TAG primers produced considerably more fragments (an average of 6.44 fragments per primer) compared with ISSR and RAPD primers (an average of 3.59 and 2.49 fragments per primer, respectively). These results indicate that the HFO-TAG primers should be more effective than ISSR or RAPD primers in targeting active gene loci. The extensive EST database available for a large number of plant and animal species should be a useful source for developing HFO-TAG primers that can be used in genetic mapping and phylogenic studies of important crop plants and animal species.

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Chubarov, Alexey S., Igor P. Oscorbin, Lidiya M. Novikova, Maxim L. Filipenko, Alexander A. Lomzov, and Dmitrii V. Pyshnyi. "Allele-Specific PCR for PIK3CA Mutation Detection Using Phosphoryl Guanidine Modified Primers." Diagnostics 13, no. 2 (January 9, 2023): 250. http://dx.doi.org/10.3390/diagnostics13020250.

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Phosphoryl guanidine (PG) is the novel uncharged modification of internucleotide phosphates of oligonucleotides. Incorporating PG modification into PCR primers leads to increased discrimination between wild-type and mutated DNA, providing extraordinary detection limits in an allele-specific real-time polymerase chain reaction (AS-PCR). Herein, we used PG-modification to improve the specificity of AS primers with unfavorable Pyr/Pur primer’s 3′-end mismatch in the template/primer complex. Two mutations of the PIK3CA gene (E542K, E545K) were chosen to validate the advantages of the PG modification. Several primers with PG modifications were synthesized for each mutation and assessed using AS-PCR with the plasmid controls and DNA obtained from formalin-fixed paraffin-embedded (FFPE) tissues. The assay allows the detection of 0.5% of mutated DNA on the wild-type DNA plasmid template′s background with good specificity. Compared with ddPCR, the primers with PG-modification demonstrated 100% specificity and 100% sensitivity on the DNA from FFPE with mutation presence higher than 0.5%. Our results indicate the high potential of PG-modified primers for point mutation detection. The main principle of the developed methodology can be used to improve the specificity of primers regardless of sequences.

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Jackson, Roy A. "Prime primers." Philosophers' Magazine, no. 8 (1999): 51. http://dx.doi.org/10.5840/tpm1999823.

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Weitzman, Jonathan B. "Modified primers." Genome Biology 3 (2002): spotlight—20020701–01. http://dx.doi.org/10.1186/gb-spotlight-20020701-01.

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Satterfield, Brent C. "Cooperative Primers." Journal of Molecular Diagnostics 16, no. 2 (March 2014): 163–73. http://dx.doi.org/10.1016/j.jmoldx.2013.10.004.

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Akhmetzianova, L. U., T. M. Davletkulov, I. M. Gubaidullin, and A. R. Islamgulov. "Parallel implementation of the primer search algorithm for loop-mediated isothermal amplification." Journal of Physics: Conference Series 2131, no. 2 (December 1, 2021): 022004. http://dx.doi.org/10.1088/1742-6596/2131/2/022004.

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Abstract In the paper, the implementation of an algorithm of search for primers in a DNA sequence with a size varying between one nucleotide and multiple million nucleotides was discussed. This analysis was done within the objective of finding a set of six specific primers that are used for a conduction of the loop-mediated isothermal amplification (LAMP). For the fastest search result possible, a parallel search for the primer with the use of the Rabin-Karp Algorithm which enables the search for a primer’s entry in DNA sequence in each thread was proposed. A new software for search for primers was developed using Python with BioPython library which implements the algorithm.

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Tsurumi, T. "Primer terminus recognition and highly processive replication by Epstein-Barr virus DNA polymerase." Biochemical Journal 280, no. 3 (December 15, 1991): 703–8. http://dx.doi.org/10.1042/bj2800703.

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The Epstein-Barr virus (EBV) DNA polymerase is essential for viral DNA replication in the lytic phase of the EBV life cycle. It efficiently extends RNA primers on the template DNA, suggesting the possible involvement of the EBV DNA polymerase in synthesizing Okazaki fragments from RNA primers on the lagging strand template. Competition experiments revealed that the EBV DNA polymerase had significantly higher affinity for primer termini hybridized to the template DNA than for the single-stranded DNA template or the single-stranded primer itself. ATP was not required either for primer terminus recognition or for sustainment of polymerization. The stimulation of the enzyme by (NH4)2SO4 was dependent on the template/primers utilized. These observations suggest that the primary and secondary structure of the template/primers are important factors for primer terminus recognition by the EBV DNA polymerase. The enzyme elongated synthetic RNA primer annealed to circular single-stranded M13 DNA coated with Escherichia coli single-stranded DNA-binding protein without dissociation. The processivity of the EBV DNA polymerase was strikingly high (greater than 7200 nucleotides) and the rate of polymerization was 12 nucleotides/s per polymerase molecule. The high processing capacity is a desirable feature in the synthesis of multiple copies of the EBV genome in rolling-circle DNA replication.

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Naqib, Ankur, Trisha Jeon, Kevin Kunstman, Weihua Wang, Yiding Shen, Dagmar Sweeney, Marieta Hyde, and Stefan J. Green. "PCR effects of melting temperature adjustment of individual primers in degenerate primer pools." PeerJ 7 (March 4, 2019): e6570. http://dx.doi.org/10.7717/peerj.6570.

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Deep sequencing of small subunit ribosomal RNA (SSU rRNA) gene amplicons continues to be the most common approach for characterization of complex microbial communities. PCR amplifications of conserved regions of SSU rRNA genes often employ degenerate pools of primers to enable targeting of a broad spectrum of organisms. One little noticed feature of such degenerate primer sets is the potential for a wide range of melting temperatures between the primer variants. The melting temperature variation of primers in a degenerate pool could lead to variable amplification efficiencies and PCR bias. Thus, we sought to adjust the melting temperature of each primer variant individually. Individual primer modifications were used to reduce theoretical melting temperature variation between primers, as well as to introduce inter-cluster nucleotide diversity during Illumina sequencing of primer regions. We demonstrate here the suitability of such primers for microbial community analysis. However, no substantial differences in microbial community structure were revealed when using primers with adjusted melting temperatures, though the optimal annealing temperature decreased.

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Kumari, Rima, Pankaj Kumar, V. K. Sharma, and Harsh Kumar. "Genome profiling of differential salt stress responsive landraces and varieties of rice using ISSR markers." Genetika 52, no. 3 (2020): 1215–33. http://dx.doi.org/10.2298/gensr2003215k.

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Using 14 ISSR primers for molecular profiling in relation to salinity tolerance of 18 landraces and varieties of rice, altogether 483 allelic variants including 236 shared and 247 unique alleles were generated with an average of 34.50 alleles per primer, revealing ample genetic differentiation and divergence amongst the entries under evaluation. Every primer generated polymorphic amplified products, but only 12 out of 14 primers yielded unique products. The primers having (AG)8YT, (CT)8A, (AG)8YA, (GA)8YT, (GA)8YC, (CT)8G, (TC)8C, (GATA)4 and (GA)8YG repeat motifs recorded relatively higher polymorphism per cent expressed in terms of the percentage of unique alleles in descending order of magnitude. Polymorphism information content of the primers varied from 0.612 to 0.992 for the primers (GACA)4 and (AG)8YA, respectively, with an average of 0.919 across the primers. Comparatively higher numerical values were obtained in respect of the primers (GA)8C, (CT)8A, (CT)8G, (TC)8C, (TC)8G, (AG)8YT, (AG)8YA, (GA)8YT, (GA)8YC, (GA)8YG and (GATA)4 amongst all the primers, reflecting their greater allelic richness and diversity. Poly-GA containing anchored primers produced the highest number (44.8) of allelic variants per primer followed by poly-CT, poly-AG and poly-TC containing anchored primers. But, the highest mean polymorphism per cent, highlighting the proportion of unique alleles, was exhibited by poly-AG followed by poly-GA, poly-CT and poly-TC containing anchored primers Clustering based on only poly-GA and poly-AG containing anchored primers provided more efficient genotypic discrimination in relation to salt stress responsiveness of the rice varieties. Moreover, a panel of only three poly-AG containing anchored primers facilitated perfect discrimination of rice varieties in accordance with their responsiveness to salinity stress. These primers can be efficiently utilized as functional instruments for connecting genotypic and phenotypic differences in relation to salt stress responsiveness. Principal coordinate analysis completely supported the results obtained from hierarchical classification of the landraces and varieties.

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Franck, Alan R., Bruce J. Cochrane, and James R. Garey. "Low-copy nuclear primers and ycf1 primers in Cactaceae." American Journal of Botany 99, no. 10 (October 2012): e405-e407. http://dx.doi.org/10.3732/ajb.1200128.

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Sozinova, O. I., N. A. Kozub, I. A. Sozinov, and Ya B. Blume. "Genome specificity of primers to puroindoline genes." Faktori eksperimental'noi evolucii organizmiv 22 (September 9, 2018): 191–96. http://dx.doi.org/10.7124/feeo.v22.947.

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Aim. Genome specificity of primers for amplification of complete coding sequences of puroindoline genes was studied. Methods. PCR with gene-specific primers was performed for amplification of puroindoline genes of wheat and related species. Results. The primer pair designated PinaDH is not gene-specific as it yields two products of amplification of the genes Pina-1 and Gsp-1. This primers pair is not specific for the Pina-1 gene of the genome V of D. villosum. The primer pairs designated PinaCM and Pinb are gene-specific as they produce one amplification product of respective length. We have demonstrated that the PinaCM primer pair is also specific for the genomes D, E and V, and the Pinb primer pair is not specific for the genome V. Conclusions. Gene and genomic specificity of primers for puroindoline genes was refined. Primers suitable for further direct sequencing of coding parts of puroindoline genes of wheat and related species were chosen. Keywords: puroindoline, Triticum aestivum, Aegilops, Dasypyrum, genome specificity.

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Liu, Liwang, Guang Liu, Yiqin Gong, Wenhao Dai, Yan Wang, Fanmin Yu, and Yunying Ren. "Evaluation of Genetic Purity of F1 Hybrid Seeds in Cabbage with RAPD, ISSR, SRAP, and SSR Markers." HortScience 42, no. 3 (June 2007): 724–27. http://dx.doi.org/10.21273/hortsci.42.3.724.

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Four molecular marker systems—RAPD (random amplified polymorphic DNA), ISSR (intersimple sequence repeat), SRAP (sequence-related amplified polymorphism), and SSR (simple sequence repeat)—were used to evaluate seed genetic purity of a hybrid cabbage cultivar ‘Zaoxia 16’. Genetic relationships of the F1 hybrids and their parents were analyzed with 157 RAPD primers, 54 ISSR primers, 84 SRAP primer combinations, and 44 SSR primers. Three RAPD primers (NAURP2006, NAURP2020, and NAURP2031), two ISSR primers (NAUISR1058 and NAUISR1062), one SRAP primer combination (NAUSR04/NAURS05), and two SSR primers (NAUSSR1011 and NAUSSR1031), which produced male and female parent-specific markers simultaneously, were selected for testing the genetic purity of the F1 seeds. A total of 210 ‘Zaoxia 16’ hybrid individuals were investigated with these eight selected primers. Of these, 12 appeared to be false hybrids. Nine of the 12 putative false hybrids, confirmed with all eight primers, exhibited similar banding patterns to the female parent, suggesting that they could be derived from selfing of the female parent. The results were in accordance with those from field evaluations. This study showed that RAPD, ISSR, SRAP, and SSR markers are highly efficient and reproducible for genetic purity testing of cabbage commercial hybrid seeds.

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Jennings, W. Bryan, Piero A. Ruschi, Gustavo Ferraro, Carla Christie Quijada, Ana Cecilia Gomes Silva-Malanski, Francisco Prosdocimi, and Paulo A. Buckup. "Barcoding the Neotropical freshwater fish fauna using a new pair of universal COI primers with a discussion of primer dimers and M13 primer tails." Genome 62, no. 2 (February 2019): 77–83. http://dx.doi.org/10.1139/gen-2018-0145.

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Designing primers for DNA barcoding is a significant challenge for the rich Neotropical fish fauna, which is comprised of ∼6000 species. Previously, researchers required multiple pairs of PCR primers or primer cocktails to obtain standard COI (i.e., mitochondrial cytochrome c oxidase subunit I) barcode sequences from assemblages of freshwater fish in this region. To simplify DNA barcoding and metabarcoding studies of Neotropical freshwater fish, we present a new pair of COI primers, which have yielded high quality barcodes across six teleost orders—Characiformes, Cichliformes, Cyprinodontiformes, Gymnotiformes, Siluriformes, and Synbranchiformes—native to South America. Following previous fish barcoding studies, we also tailed our primers with M13 forward and reverse primers to facilitate the DNA sequencing process. Although this practice generates primer dimers, we obtained complete and high quality COI barcode sequences for all samples. We discuss the problem of primer dimers and suggest strategies for neutralizing their influence on data quality.

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Al Khazraji, Haidar A. K., Abdulkareem M. Abd, and Abdulla A. Abdulla. "The Determination of the Genetic Distance of Various Snake Melon Cucumis melo var. flexuosus Cultivars Using Inter Simple Sequence Repeat Technique (ISSR)." Basrah J. Agric. Sci. 34, no. 1 (February 19, 2021): 111–23. http://dx.doi.org/10.37077/25200860.2021.34.1.10.

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The experiment have been done on the winter farming season 2020 in one of the farms that belongs to Faris company in Basrah governorate, the technique ISSR have been used to study the genetic distance for twenty one isolates of snake melon Cucumis melo var. flexuosus species. The variations between amplified samples have been revealed after running them on a gel of agarose which have been previously stained by ethidium bromide. Five primers which gave varied product on the agarose have been selected. Those five primers produced 713 bands, both primers UBC 813 and UBC 815 showed the higher numbers of bands reached to 177 while the primer UBC862 showed the least numbers of bands (100) and the bands which showed multi-variations showed (46) bands, and the results primers amplification unique bands their number reached to (14) and five of those bands belong to the primer UBC842 while the primer UBC862 produced three bands while the primer UBC807 did not produced any bands. While it has shown 100% polymorphisms with the primers 813, 815, 842 and 862, and the least polymorphism percentage have shown with the primer UBC807 reached 75%. According to the efficiency primers, the highest efficiency percentage shown with the primer UBC813 and 815 reached to 24.82% and the least percentage shown 14.02% by the primer UBC862. Cluster analysis showed the effect on the variance of the studied cultivars.

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Khaira, Annisa, Afifatul Achyar, Zulyusri Zulyusri, Yusni Atifah, Dwi Hilda Putri, and Violita Violita. "Primer Design and Optimization of Annealing Temperature for Analysis of Glutathione Reductase Gene Expression in Rice (Oryza sativa L.)." 3BIO: Journal of Biological Science, Technology and Management 5, no. 1 (November 30, 2023): 142–48. http://dx.doi.org/10.5614/3bio.2023.5.1.3.

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Glutathione Reductase (GR) belongs to the NADPH-dependent flavoprotein oxidoreductase family and is found in both prokaryotes and eukaryotes. The GR gene is considered to play a key role in the elimination of oxidative reaction products by looking at the level of gene expression of GR rice in dealing with drought stress using qPCR. One of the important steps to develop a specific, effective and efficient qPCR is the primer design. Several studies analyzing GR gene expression in rice have also designed primers. However, the primer still lacks an ideal characteristic of primer, as it still has a secondary structure. This studies aims to design rice GR specific primers and optimize the annealing temperature for GR gene expression analysis on rice. Primers were designed using the Primer3 and Geneious Prime and checked for specificity using the Primer-BLAST tool. The selected primer pairs were then optimized for annealing temperature using gradient PCR. The best primer design results were GR-Forward 5’-ACGATTGCAGCCAGTGAAGA-3’ and GR-Reverse 5’-TGCGGCAATACTATCAACATCC-3’, with an amplicon length of 204 bp, primer base lengths of 20 and 22 nucleotides, Tm values of 60°C and 58.9°C, %GC of 50% and 45.5%, respectively. This primer pair had no secondary structure, both hairpin and self dimer. Gradient PCR showed the optimum annealing temperature for this primer pair was 52.2oC so that the primer can be used as a specific primer to analyze the GR gene expression in rice using qPCR.

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Ismail, Aisyah Mohd, and Farida Zuraina Mohd Yusof. "The Efficiency of Long Primers Compared to The Short Primer for RAPD Technique in Date Palm." Science Letters 16, no. 1 (January 9, 2022): 1. http://dx.doi.org/10.24191/sl.v16i1.13444.

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Random Amplified Polymorphic DNA (RAPD) applies single arbitrary short primers (8-12nucleotides) to produce many amplified discrete DNA. Limited reports and studies were done onthe use of long primers (over 12 bases). This study was performed to investigate the potential valueof long primers (15-21 bases) for generating RAPD polymorphisms. We compared both short andlong primers in RAPD assays of two date palm cultivars grown in Malaysia: Ajwa and Barhi. Thenumber of produced polymorphic fragments ranged in order from 2 and 38 bands for short andlong primers in Ajwa. Meanwhile, more polymorphic fragments were generated by long primersin Barhi, which were 50 and only five bands for short primers. 18-mer GY107 and 20-mer CO4primers yielded 100% polymorphism in Ajwa and Barhi, respectively. Moreover, long primersproduced more DNA fragments and a wider range of DNA fragment sizes (from 140-1600 bp,with respect to 300-1000 bp obtained with 10-mer primers). Hence, a significant correlation wasobserved between primer length and the number of polymorphic fragments within the long primergroup, suggesting that increasing primer length above 15 bases may demonstrate enhancedproduction of more polymorphism.

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Ravindran, Aravind, Julien Levy, Elizabeth Pierson, and Dennis C. Gross. "Development of Primers for Improved PCR Detection of the Potato Zebra Chip Pathogen, ‘Candidatus Liberibacter solanacearum’." Plant Disease 95, no. 12 (December 2011): 1542–46. http://dx.doi.org/10.1094/pdis-05-11-0386.

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Zebra chip disease poses a major economic threat to potato production. The causative agent is a phloem-limited bacterium identified as ‘Candidatus Liberibacter solanacearum’ that is transmitted by the potato/ tomato psyllid. Currently, there are no effective controls and existing control strategies depend largely on the early detection of the pathogen via polymerase chain reaction (PCR) assays. Most primer sets used for PCR detection target a region of the bacterial 16S rDNA gene, and detection of the pathogen in symptomatic potato tissue with existing primers has been variable depending on the specific primer sets used. This study describes the development of two new primer sets that target a conserved intergenic region between the 16S and 23S rDNA genes and a conserved bacterial housekeeping gene, adenylate kinase (adk). Results demonstrate that the new primer sets are more reliable in detecting ‘Ca. L. solanacearum’ in field and glasshouse samples than the currently used LsoF/OI2 primers. The newly developed primers differentiated between ‘Ca. L. solanacearum’ and a closely related ‘Ca. Liberibacter’ spp. and were more sensitive than the LsoF/OI2 primers. The low detection limit for the new primers was four times lower (0.65 ng) than the limit (2.5 ng) for the LsoF/OI2 primers.

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Lall, Sabbi. "Primers on chromatin." Nature Structural & Molecular Biology 14, no. 11 (November 2007): 1110–15. http://dx.doi.org/10.1038/nsmb1107-1110.

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Na, Hee Sam, Yuri Song, Yeuni Yu, and Jin Chung. "Comparative Analysis of Primers Used for 16S rRNA Gene Sequencing in Oral Microbiome Studies." Methods and Protocols 6, no. 4 (August 6, 2023): 71. http://dx.doi.org/10.3390/mps6040071.

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Recent advances in genomic technologies have enabled more in-depth study of the oral microbiome. In this study, we compared the amplicons generated by primers targeting different sites of the 16S rRNA gene found in the Human Oral Microbiome Database (HOMD). Six sets of primer targeting V1–V2, V1–V3, V3–V4, V4–V5, V5–V7 and V6–V8 regions of 16S rRNA were tested via in silico simulation. Primers targeting the V1–V2, V3–V4, and V4–V5 regions generated more than 90% of the original input sequences. Primers targeting the V1–V2 and V1–V3 regions exhibited a low number of mismatches and unclassified sequences at the taxonomic level, but there were notable discrepancies at the species level. Phylogenetic tree comparisons showed primers targeting the V1–V2 and V3–V4 regions showed performances similar to primers targeting the whole 16s RNA region in terms of separating total oral microbiomes and periodontopathogens. In an analysis of clinical oral samples, V1–V2 primers showed superior performance for identifying more taxa and had better resolution sensitivity for Streptococcus than V3–V4 primers. In conclusion, primers targeting the V1–V2 region of 16S rRNA showed the best performance for oral microbiome studies. In addition, the study demonstrates the need for careful PCR primer selections.

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Abdullah, Asadatun, Annisaa Putri, and Mala Nurilmala. "Specific Primer Design for Detection of gene Cyt b for Shark Species Prionace glauca and gene COI for Carcharhinus spp. Using Real-Time PCR Method." BIO Web of Conferences 92 (2024): 01008. http://dx.doi.org/10.1051/bioconf/20249201008.

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International market demand for sharks is increasing, thus increasing the number of catches of several shark species that are included in the International Union for Conservation of Nature’s (IUCN) Red List and the Convention International Trade of Endangered Species (CITES) Appendix II. Authentication using biomoleculars by utilizing DNA is necessary. This study was aimed to design specific primers based on cytochrome c oxidase I and cytochrome b marker genes for endangered sharks (Prionace glauca and Carcharhinus spp.) and to apply them in vitro to identify fishery products using real-time PCR techniques. This research begins with designing target species-specific primers. Designing a specific primer using BioEdit software. The Oligo Evaluator and NCBI’s Blast (Basic Local Alignment Search Tool) web tool for performing primary specification validation. The next stage is the sample preparation, DNA isolation, DNA amplification, DNA quality and quantity testing, and realtime PCR analysis. Primer’s design of target species Prionace glauca using cyt b gene and degenerate primer for genus Carcharhinus spp. COI gene markers were successfully carried out in silico. Efficient real-time PCR conditions of Prionace glauca and Carcharhinus spp. complies with testing standards using the real-time PCR method.

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Parent, Jean-Guy, and Daniele Page. "Evaluation of SCAR Markers to Identify Raspberry Cultivars." HortScience 30, no. 4 (July 1995): 856B—856. http://dx.doi.org/10.21273/hortsci.30.4.856b.

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Random amplified polymorphic DNA (RAPD) markers are used in Quebec's certification program to verify the identity of raspberry cultivars. However, sequence characterized amplified region (SCAR) markers, less sensitive to modifications in reaction conditions, could be derived from RAPD markers. Our objective was to evaluate the potential of SCAR markers to replace the RAPD ones. Five RAPD markers obtained with primer OPG06 (length of 520, 700, 825, 1450, and 2000 bp) were cloned in pTZ/PC or pCRII vectors. Extremities of the cloned markers were sequenced by the nonradioactive silver sequence method using pUC/M13 forward and reverse primers. Sequence information was used to make SCAR primers, similar in length to standard PCR primers. Some SCAR primers were elongated RAPD primers, whereas others were from internal regions. Ability of primer pairs and combination of primer pairs to discriminate cultivars of our certification program was compared with their RAPD counterparts as well as with the technical feasibility of both methods.

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Journal, Baghdad Science. "Use of DAF markers (DNA Amplification Fingerprint) to Assess Genetic Diversity of Rice (Oryza satival L.)." Baghdad Science Journal 6, no. 2 (June 7, 2009): 179–289. http://dx.doi.org/10.21123/bsj.6.2.179-289.

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This study was carried out to assess genetic diversity of ten cultivars of Rice (Oryza sativa L.). One of DNA markers based on Polymerase Chain Reaction (PCR) was used namely DAF markers (DNA Amplification Fingerprint). Six primers were tested, the results showed, that no amplification products using the primers OPD.14 and OPM.5. Two primers (OPX.8 and OPT.2) produced monomorphic band across all cultivars, while only two primers generated polymorphic bands. The number of total bands produced from one of them (OPN.7) were sixteen. Also this primer produced ten polymorphic profiles (DAF patterns) which were unique to the ten cultivars that could be distinguished. The number of total bands generated by primer OPX.1 were thirteen and this primer produced eight polymorphic patterns which was unique for distinguishing six cultivars. This means that DAF markers were able to identify all rice cultivars using only two primers reflecting the high potentialities of these markers for their applications in fingerprinting.

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Wu, Yueni, Kai Feng, Ziyan Wei, Zhujun Wang, and Ye Deng. "ARDEP, a Rapid Degenerate Primer Design Pipeline Based on k-mers for Amplicon Microbiome Studies." International Journal of Environmental Research and Public Health 17, no. 16 (August 17, 2020): 5958. http://dx.doi.org/10.3390/ijerph17165958.

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The survey of microbial diversity in various environments has relied upon the widespread use of well-evaluated amplification primers for taxonomic marker genes (e.g., prokaryotic 16S and fungal ITS). However, it is urgent to develop a fast and accurate bioinformatic program to design primers for microbial functional genes to explore more mechanisms in the microbial community. Here, we provide a rapid degenerate primer design pipeline (ARDEP) based on the k-mer algorithm, which can bypass the time-consuming step of sequence alignment to greatly reduce run times while ensuring accuracy. In addition, we developed an open-access platform for the implementation of primer design projects that could also calculate the amplification product length, GC content, Annealing Temperature (Tm), and ΔG of primer self-folding, and identify covered species and functional groups. Using this new platform, we designed primers for several functional genes in the nitrogen cycle, including napA and amoA. Our newly designed primers achieved higher coverage than the commonly used primers for all tested genes. The program and the associated platform that applied the k-mer algorithm could greatly enhance the design and evaluation of primers for environmental microbiome studies.

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Dederichs, Marco, Zaid Badr, Stephanie Viebranz, Steffen Schroeter, Christoph-Ludwig Hennig, Anne-Sophie Schmelzer, and Arndt Guentsch. "Effect of Different Primers on Shear Bond Strength of Base Metal Alloys and Zirconia Frameworks." Polymers 16, no. 5 (February 20, 2024): 572. http://dx.doi.org/10.3390/polym16050572.

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Ensuring a secure bond between a framework structure and layering composite resin veneer is essential for a long-lasting dental restoration. A variety of primer systems are available to facilitate the adhesive bonding. Nevertheless, the growing preference for efficiency and simplicity in dentistry has made the one-bottle universal primers a desirable option. This study aims to compare the effectiveness of universal primers on the shear bond strength (SBS) of base metal alloy (BMA) and zirconia to layering composite resin. Each 160 BMA and zirconia 20 × 10 × 5 mm test specimen was fabricated. Eight different primers (SunCera Metal Primer, Metal Primer Z, Reliance Metal Primer, Alloy Primer, MKZ Primer, Monobond Plus, ArtPrime Plus, and Clearfil Ceramic Primer Plus) were applied to 20 specimens in each group. Subsequently, a 5 × 2 mm composite resin build-up was applied. SBS tests were performed after 24 h of water storage and after thermocycling (25,000 cycles, 5–55 °C). On BMA, after water storage for 24 h, the bond strength values ranged from 26.53 ± 3.28 MPa (Metal Primer Z) to 29.72 ± 2.00 MPa (MKZ Primer), while after thermocycling, bond strength values ranged from 25.19 ± 1.73 MPa (MKZ Primer) to 27.69 ± 2.37 MPa (Clearfil Ceramic Primer Plus). On a zirconia base, after 24 h, the bond strengths values ranged from 22.63 ± 2.28 MPa (Reliance Primer) to 29.96 ± 2.37 MPa (MKZ Primer) and from 23.77 ± 3.86 MPa (Metal Primer Z) to 28.88 ± 3.09 MPa (Monobond Plus) after thermocycling. While no significant difference in bond strength was found between the primers on the BMA base, five primer combinations differed significantly from each other on zirconia (p = 0.002–0.043). All primers achieved a bond strength greater than 23 MPa on both framework materials after thermocycling. Thus, all primers tested can be applied to both framework materials with comparable results.

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Lundgaard, Stefan, Soon Ng, Damien Cahill, Johan Dahlberg, Dong Ruan, Nerida Cole, Paul Stoddart, and Saulius Juodkazis. "Towards Safer Primers: A Review." Technologies 7, no. 4 (October 18, 2019): 75. http://dx.doi.org/10.3390/technologies7040075.

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

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Scibetta, Silvia, Leonardo Schena, Ahmed Abdelfattah, Sonia Pangallo, and Santa O. Cacciola. "Selection and Experimental Evaluation of Universal Primers to Study the Fungal Microbiome of Higher Plants." Phytobiomes Journal 2, no. 4 (January 2018): 225–36. http://dx.doi.org/10.1094/pbiomes-02-18-0009-r.

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The impact of primer choice on results of metabarcoding studies was experimentally evaluated by analyzing fungal communities associated with leaves of four plant species. Significant differences in target specificity of primers were highlighted by a percentage of plant reads ranging from almost nothing to 30 to 35% of the total detected sequences. Overall, primer sets targeting the internal transcribed spacer 1 (ITS1) region proved to be more specific than those targeting the ITS2 region. A comparable taxa coverage was revealed for all investigated primer sets. However, each primer set detected only around 50% of the overall detected taxa highlighting that a consistent part of the actual fungal diversity remains undetected in studies conducted using a single couple of primers. The coverage was increased to 70 to 80% by combining results from two different primer sets. Some fungal taxa were preferentially or exclusively detected by certain primer sets and this association between primers and taxa was generally recurrent on several plant hosts. Data highlighted that a perfect set of primers to investigate the whole fungal diversity does not exist and that whatever the choice, only a fraction of the actual microbial diversity will be investigated. However, provided information may be valuable to select the best primers according to the objective of the analysis.

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Hu, Ying, Li Bai, Linna Zhao, Lingling Wu, Hong Lv, Qiongqiong Li, Xinpeng Li, et al. "Standardized Shiga-Toxin Encoding Genes Real-Time PCR Screening Methods Comparison and Development of an Internally Controlled Assay for Pan-stx2 Detection." Journal of AOAC INTERNATIONAL 104, no. 4 (March 16, 2021): 1065–71. http://dx.doi.org/10.1093/jaoacint/qsab030.

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Abstract Background Various primer and probe sets have been developed and standardized, but certain sets may have low efficiency or miss some stx-subtypes. Objective To compare the efficiency of the recommended stx screening primers and probe sets in four standardized methods and develop a new primers and probe system with an internal amplification control (IAC) for all known stx2 subtypes. Method The inclusivity and specificity of recommended screening primers and probe sets in four standardized methods were compared. A new pan-stx2 primer and probe set was adapted from the International Organization for Standardization (ISO) method for all known stx2 subtypes. The robustness of the new method was assessed in seven laboratories and also assessed in ground beef and bean sprout samples. Results None of the recommended screening primers and probe sets in the four standardized methods could efficiently amplify all the stx2 subtypes because of various mismatches in the primers or the probe sequences. A new primers and probe system adapted from the ISO method, through introducing degenerate bases in primers and probe sequences with an IAC, showed high amplification efficiency and specificity for all known stx2 subtypes in ground beef and bean sprouts samples. The specificity of the new method was assessed in seven laboratories and showed robust and consistent results. Conclusions This study provided evidence for Shiga-toxin producing Escherichia coli (STEC) screening method development, and the newly developed primers and probes system should be considered in the revision of the standardized methods. Highlights None of the recommended screening primer and probe set in the four official methods could efficiently amplify all the stx2 subtypes. A new developed primer and probe set showed high amplification efficiency and specificity for all known stx2 subtypes in fresh ground beef and bean sprouts samples. The newly developed stx2 screening system showed robustness and consistency during interlaboratory study.

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Barak, Noga, Eduard Fadeev, Vera Brekhman, Dikla Aharonovich, Tamar Lotan, and Daniel Sher. "Selecting 16S rRNA Primers for Microbiome Analysis in a Host–Microbe System: The Case of the Jellyfish Rhopilema nomadica." Microorganisms 11, no. 4 (April 6, 2023): 955. http://dx.doi.org/10.3390/microorganisms11040955.

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Amplicon sequencing of the 16S rRNA gene is extensively used to characterize bacterial communities, including those living in association with eukaryotic hosts. Deciding which region of the 16S rRNA gene to analyze and selecting the appropriate PCR primers remains a major decision when initiating any new microbiome study. Based on a detailed literature survey of studies focusing on cnidarian microbiomes, we compared three commonly used primers targeting different hypervariable regions of the 16S rRNA gene, V1V2, V3V4, and V4V5, using the jellyfish Rhopilema nomadica as a model. Although all primers exhibit a similar pattern in bacterial community composition, the performance of the V3V4 primer set was superior to V1V2 and V4V5. The V1V2 primers misclassified bacteria from the Bacilli class and exhibited low classification resolution for Rickettsiales, which represent the second most abundant 16S rRNA gene sequence in all the primers. The V4V5 primer set detected almost the same community composition as the V3V4, but the ability of these primers to also amplify the eukaryotic 18S rRNA gene may hinder bacterial community observations. However, after overcoming the challenges possessed by each one of those primers, we found that all three of them show very similar bacterial community dynamics and compositions. Nevertheless, based on our results, we propose that the V3V4 primer set is potentially the most suitable for studying jellyfish-associated bacterial communities. Our results suggest that, at least for jellyfish samples, it may be feasible to directly compare microbial community estimates from different studies, each using different primers but otherwise similar experimental protocols. More generally, we recommend specifically testing different primers for each new organism or system as a prelude to large-scale 16S rRNA gene amplicon analyses, especially of previously unstudied host–microbe associations.

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De Paula, DM, AD Loguercio, A. Reis, S. Sauro, AH Alves, PR Picanço, K. Yoshihara, and VP Feitosa. "Lack of Neutralization of 10-MDP Primers by Zirconia May Affect the Degree of Conversion of Dual-cure Resin Cement." Operative Dentistry 46, no. 1 (January 1, 2021): 107–15. http://dx.doi.org/10.2341/18-189-l.

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Clinical Relevance Use of zirconia primers with a low pH and a high acidic monomer concentration should be employed in combination with dual-cure resin cements that are less sensitive to an acidic environment. Primers with lower 10-MDP concentrations attain better outcomes. SUMMARY Objective: To assess the effects of different concentrations of 10-methacryloyloxydecyl dihydrogen phosphate (10-MDP) included in experimental ceramic primers on the degree of conversion (DC) and microshear bond strength (μSBS) of a dual-cure resin cement, and on the acidity neutralization potential of zirconia (ZrO2) in comparison to hydroxyapatite (HAp). Methods: Experimental ceramic primers were formulated using 5 wt%, 10 wt%, 20 wt%, or 40 wt% 10-MDP as an acidic functional monomer and camphorquinone (CQ)/amine or 1-phenyl-1,2-propanedione (PPD) as a photoinitiator system. Clearfil Ceramic Primer (Kuraray Dental, Tokyo, Japan) was used as the commercial control. Micro-Raman spectroscopy was used to assess the DC of uncured and light-cured resin cements applied onto primer-treated ZrO2 surfaces. The μSBS and pH of primers were assayed in a universal testing machine and by a digital pH meter (Tec-3MP; Tecnal, Piracicaba, Brazil), respectively. Statistical analysis was performed by one-way analysis of variance (ANOVA) and Tukey’s test (p<0.05). Results: DC was not affected until a concentration of 10% 10-MDP in CQ primer and 5% 10-MDP in PPD primer was reached, when compared with the positive control (p>0.05). Groups 10-MDP 5% in CQ and PPD primers showed the highest μSBS compared with the positive control (p>0.05); however, higher concentrations of 10-MDP induced significant DC and μSBS reduction (p<0.05). HAp neutralized 10-MDP primers, but ZrO2 provided higher acidity to the primers’ pH. Conclusion: 10-MDP monomer should be used in low concentrations in ZrO2 primers to avoid reduction of the polymerization and bond strength of resin cement.

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Martins, B. C., D. M. De Paula, A. D. Loguercio, A. Reis, and V. P. Feitosa. "A Falta de Neutralização de Primers com 10-MDP pela Zircônia Pode Afetar no Grau de Conversão do Cimento Resinoso." Journal of Health Sciences 19, no. 5 (February 23, 2018): 92. http://dx.doi.org/10.17921/2447-8938.2017v19n5p92.

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O objetivo deste trabalho foi avaliar diferentes concentrações de 10-MDP incluídas em primers cerâmicos experimentais e comerciais e o efeito sobre o grau de conversão (GC) de um cimento resinoso dual convencional. Os primers cerâmicos experimentais foram formulados utilizando o10-metacriloiloxi-decil-dihidrogenofosfato (10-MDP) nas concentrações 5, 10, 20 e 40% como monômero funcional ácido e canforoquinona /amina terciária (CQ) ou 1-fenil-1,2-propanodiona (PPD) como sistemas fotoiniciadores. O primer cerâmico Clearfil Ceramic Primer (Kuraray) foi utilizado como primer controle comercial. O pH dos primers foi analisado com medidor digital de pH. A espectroscopia Micro-Raman foi usada para avaliar o GC pelas razões das alturas dos picos 1639/1609 cm-1 do cimento resinoso não-polimerizado e polimerizado aplicado após os primers cerâmicos na superfície de um bloco de zircônia. A análise estatística foi realizada por ANOVA one-way e teste de Tukey (p<0,05). A alta concentração (40%) de 10-MDP em primers experimentais não foi tamponada pela ZrO2 e reduziu a GC do cimento resinoso quando utilizado o primer com CQ (63,4 ± 4,9 %) e PPD (44,6 ± 4,2 %), o que não ocorreu com 10-MDP de baixa concentração (5%) no primer comercial (88,9 ± 1,9 %) e nos experimentais CQ (94,7±1,5 %) e PPD (92,0±3,1 %). O monômero 10-MDP deve ser utilizado em baixas concentrações em primers para zircônia para evitar a redução na polimerização do cimento resinoso. Apoio: FGM. Palavras-chave: Metacrilato. Grau de Conversão. Espectroscopia Raman. Zircônia.

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Grauke, L. J., T. E. Thompson, and A. S. Reddy. "AFLPs in Pecan Genetics Research." HortScience 33, no. 3 (June 1998): 527b—527. http://dx.doi.org/10.21273/hortsci.33.3.527b.

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Procedures were refined for extraction and amplification of DNA from pecan [Carya illinoinensis (Wangenh.) K. Koch] leaf tissue. Genomic DNA was extracted from leaf tissue from multiple inventories of `Wichita' and `Pawnee' and processed for Amplified Fragment Length Polymorphism (AFLPs). Using only four AFLP primers, 26 polymorphisms were identified, verifying the reproducibility and consistency of amplification. The application and limitation of the procedure for separating genotypes will be discussed. Twenty-four cultivars and seedlings representing the geographic range of the species were analyzed using 10 primer combinations. Despite the small sample size, polymorphic bands apparently associated with geographic origin were apparent. Individuals from selected controlled-cross families of the Pecan Breeding Program were bulked according to disease reaction and screened using 64 primers. Primary primers were selected on the basis of polymorphisms observed in bulked samples of resistant and susceptible genotypes. Eighteen primer combinations were selected for use on all individuals in the test. The candidate markers were evaluated to verify that parental lines were polymorphic for the trait, reducing to one the number of appropriate primers. That primer was used to screen 84 progeny samples phenotypically rated for disease resistance levels. The data were analyzed for linkage to scab resistance in the population. Factors limiting the utility of AFLPs as tools for selection of disease resistant genotypes, and their use in developing markers for heterodichogamy (a simple dominant genetic system) will also be discussed.

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Akhmetzianova, L. U., T. M. Davletkulov, R. R. Garafutdinov, and I. M. Gubaydullin. "Application of the Aho-Korasik Algorithm for the Selection of Primers for Loop Isothermal Amplification." Mathematical Biology and Bioinformatics 17, no. 2 (November 14, 2022): 250–65. http://dx.doi.org/10.17537/2022.17.250.

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This paper presents a program which allows user to do primer design for identifying DNA target site or a whole genome with a goal of performing loopmediated isothermal amplification. The review of the most popular existing primer design programs for LAMP is carried out. Recommended conditions are presented in the paper. They are required to be taken in consideration during the process of primer design for loop-mediated isothermal amplification. These are the conditions: primer’s length, GC-content, amplicon average size, annealing temperature and distance between primers. A search for primer positions in genome is needed since loop-mediated isothermal amplification requires primer kits that consist of 6 primers in order for primer design to be done. The Aho–Corasick algorithm was proposed for a search implementation. This algorithm is capable of simultaneous search for a number of sample (primer) entries in a longer sequence (a fragment or a whole genome). This software allows the search for primers in genomes of various length and it groups primers by kits, which in turn could be applied in laboratory experiments. These kits are formed according both to the recommended conditions of primer selection for performing loop-mediated isothermal amplification and to the initial conditions, which are determined by the user before the process. After that, the user may choose the best option for their case from a list of primer kits that are being created as a result of performed computer analysis. The test run of the program was done during the search for a specific primer kit that is meant to be used for performing loop-mediated isothermal amplification of genome with a goal of detection of novel coronavirus infection SARS-CoV-2, a virus that triggers a dangerous disease, COVID-19. The software was developed using Python with BioPython and Pyahocorasick libraries and available at the link: https://cloud.mail.ru/public/C7av/QCkSiUomz.

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Ogliari, Juliana Bernardi, Raquel L. Boscariol, and Luis E. A. Camargo. "Optimization of PCR amplification of maize microsatellite loci." Genetics and Molecular Biology 23, no. 2 (June 2000): 395–98. http://dx.doi.org/10.1590/s1415-47572000000200026.

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Maize (Zea mays L.) microsatellite loci are useful as genetic markers because they are numerous, occur in every chromosome, and have a high content of polymorphism information. Furthermore, they can be amplified by PCR and the resulting fragments resolved on agarose gels. A major problem, however, is that the primers used in their amplification often have different length and nucleic acid content, requiring optimization of PCR amplification programs for each locus. We developed "touchdown" PCR programs successfully used in the amplification of a set of 125-maize microsatellite loci, chosen as representative of all chromosomes. The programs varied both in relation to the annealing temperature (Tm) and primer concentration. Primers could be divided into four groups. A large group included primers that amplified well in a basic previously published amplification program but with different primer concentrations. A second group amplified in alternative programs in which the annealing temperatures were changed so as to include the Tm of either both primers or the highest Tm of the primer pair estimated based on their nucleotide composition. A third group included primers that amplified in programs with Tm higher than those estimated, and in a fourth group, with just two pairs of primers, the opposite situation prevailed.

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Carr, Shannon M., and Kelly M. Elkins. "Development of Polymerase Chain Reaction–High-Resolution Melt Assay for Waterborne Pathogens Legionella pneumophila, Vibrio parahaemolyticus, and Camplobacter jejuni." Microorganisms 12, no. 7 (July 3, 2024): 1366. http://dx.doi.org/10.3390/microorganisms12071366.

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Legionella pneumophila is the waterborne pathogen primarily responsible for causing both Pontiac Fever and Legionnaire’s Disease in humans. L. pneumophila is transmitted via aerosolized water droplets. The purpose of this study was to design and test primers to allow for rapid polymerase chain reaction (PCR) melt detection and identification of this infectious agent in cases of clinical or emergency response detection. New PCR primers were designed for this species of bacteria; the primer set was purchased from IDT and the target bacterial DNA was purchased from ATCC. The L. pneumophila primers targeted the macrophage infectivity potentiator gene (mip), which inhibits macrophage phagocytosis. The primers were tested for specificity, repeatability, and sensitivity using PCR–high-resolution melt (HRM) assays. The primer set was found to be specific to the designated bacteria and did not amplify the other twenty-one species from the panel. The L. pneumophila assay was able to be multiplexed. The duplex assay consists of primers for L. pneumophila and Vibrio parahaemolyticus, which are both waterborne pathogens. The triplex assay consists of primers for L. pneumophila, V. parahaemolyticus, and Campylobacter jejuni. The unique melting temperature for the L. pneumophila primer assay is 82.84 ± 0.19 °C, the C. jejuni assay is 78.10 ± 0.58 °C, and the V. parahaemolyticus assay is 86.74 ± 0.65 °C.

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Shaha, Modhusudon, Bithi Roy, and Mohammad Ariful Islam. "Detection of herpes simplex virus 2: a SYBR-Green-based real-time PCR assay." F1000Research 10 (November 9, 2021): 655. http://dx.doi.org/10.12688/f1000research.53541.2.

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The prevalence of Herpes simplex virus 2 (HSV2) is increasing at an alarming rate in the world. Most of the HSV2 cases are not diagnosed properly, although a range of molecular and serological diagnoses exist. Herein, we have reported a very rapid detection method specific for HSV2 using real-time PCR. The primers specific for HSV2 were designed using the Primer-BLAST tool and 120 base pairs of the polymerase gene were amplified using real-time PCR with SYBR Green dye. The designed primer pair was found highly efficient in detecting only HSV2 DNA, but not HSV1. The threshold cycle (Ct) value for HSV2 reactions by designed primers was found to be an average of 22.55 for a standard copy number of viral DNA that may denote the efficiency of the primers. The melting temperature (Tm) of the amplicon using designed primers (82.60C) was also higher than that using reference primers (about 780C), indicating the high GC content of the amplified template. The designed primer pair will help clinicians to detect the HSV2 DNA specifically and diagnose the associated disease rapidly.

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Shaha, Modhusudon, Bithi Roy, and Mohammad Ariful Islam. "Rapid detection of herpes simplex virus 2: a SYBR-Green-based real-time PCR assay." F1000Research 10 (July 26, 2021): 655. http://dx.doi.org/10.12688/f1000research.53541.1.

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The prevalence of Herpes simplex virus 2 (HSV2) is increasing at an alarming rate in the world. Most of the HSV2 cases are not diagnosed properly, although a range of molecular and serological diagnoses exist. Herein, we have reported a very rapid detection method specific for HSV2 using real-time PCR. The primers specific for HSV2 were designed using the Primer-BLAST tool and 120 base pairs of the polymerase gene were amplified using real-time PCR with SYBR Green dye. The designed primer pair was found highly efficient in detecting only HSV2 DNA, but not HSV1. The threshold cycle (Ct) value for HSV2 reactions by designed primers was found to be an average of 22.55 for a standard copy number of viral DNA that may denote the efficiency of the primers. The melting temperature (Tm) of the amplicon using designed primers (82.60C) was also higher than that using reference primers (about 780C), indicating the high GC content of the amplified template. The designed primer pair will help clinicians to detect the HSV2 DNA specifically and diagnose the associated disease rapidly.

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Zhou, Jing. "Polycystins and Primary Cilia: Primers for Cell Cycle Progression." Annual Review of Physiology 71, no. 1 (March 2009): 83–113. http://dx.doi.org/10.1146/annurev.physiol.70.113006.100621.

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Fu, Qi, Paul Ruegger, Elizabeth Bent, Marek Chrobak, and James Borneman. "PRISE (PRImer SElector): Software for designing sequence-selective PCR primers." Journal of Microbiological Methods 72, no. 3 (March 2008): 263–67. http://dx.doi.org/10.1016/j.mimet.2007.12.004.

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Di Francescantonio, Marina, Marcelo Tavares de Oliveira, Luiz Gustavo Dias Daroz, Guilherme Elias Pessanha Henriques, and Marcelo Giannini. "Adhesive bonding of resin cements to cast titanium with adhesive primers." Brazilian Dental Journal 23, no. 3 (2012): 218–22. http://dx.doi.org/10.1590/s0103-64402012000300006.

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The purpose of this study was to evaluate the effects of adhesive primer applications on the bond strength of resin cements to cast titanium. Four adhesive primers - Metaltite, Metal Primer II, Alloy Primer and Ceramic Primer - and their respective resin cements - Bistite II DC, Link Max, Panavia F 2.0, RelyX Unicem and RelyX ARC - were tested. Cast plates were prepared from titanium ingots (n=6 specimens/cement) and had their surfaces airborne-particle abraded with Al2O3 (50 μ m). Three resin cement cylinders were built on each bonded titanium surface, using a cylindrical translucent tubing mold and were subjected to micro-shear testing. Data were analyzed statistically by two-way ANOVA and Tukey's post-hoc test (α=0.05). The application of Metal Primer II and Ceramic Primer resulted in significant higher bond strength for Link Max and RelyX Unicem resin cements, respectively, than nonuse of adhesive primers. Panavia F 2.0 and RelyX ARC yielded high bond strength means with or without adhesive primers. The use of adhesive primers might increase the bond strength to cast titanium depending on the resin cement used.

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Krasņevska, Nikole, Andra Miķelsone, Alesia Kruchonok, Isaak Rashal, Dalius Butkauskas, and Dace Grauda. "Assessment of iPBS Primers Potential to Be Used in Genetic Diversity Studies of Wild Cloudberry (Rubus Chamaemorus L.) Populations." Proceedings of the Latvian Academy of Sciences. Section B. Natural, Exact, and Applied Sciences. 76, no. 2 (April 1, 2022): 314–16. http://dx.doi.org/10.2478/prolas-2022-0045.

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Abstract Inter-primer binding site (iPBS) molecular retrotransposon-based markers are a universal and simple tool to estimate genetic diversity. The goal of this study was to select highly informative iPBS primers to be used in studies of intraspecific genetic diversity of cloudberry (Rubus chamaemorus L.), a common plant species of the boreal zone, associated with raised bog habitats and transitional bogs. Initially, 75 iPBS primers were screened and three of the most informative primers (indicated by numbers 2298, 2277, and 2229) were selected based on the highest number of polymorphic bands and PIC (polymorphic information content) values. Electrophoretic analysis revealed 34 bands amplified with primer 2229, 25 bands with primer 2298, and 22 bands with primer 2277. The percentage of polymorphic loci ranged from 90.9 to 97.1% and PIC values ranged from 0.4885 to 0.4965, indicating potential of the selected primers to be applied in population genetic studies of R. chamaemorus.

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Joshi, Bal K., Kazutoshi Okuno, Ryo Ohsawa, and Takashi Hara. "Optimization of PCR Conditions for DNA Amplification of Common Buckwheat Using EST Primers." Nepal Agriculture Research Journal 8 (November 18, 2014): 1–6. http://dx.doi.org/10.3126/narj.v8i0.11563.

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Under optimal conditions the PCR reaction is very efficient; microgram quantities may be synthesized from a single molecule of substrate DNA. DNA of four lines of common buckwheat (Kyusu, Canada, Miyazaki and Botansoba) was used to optimize PCR reaction and cycling program of 26 primers for DNA amplification of common buckwheat. Annealing temperature (Ta), PCR cycle number and MgCl2 concentration were considered optimum if the single clear band was observed. Of the 26 primers Ta of only 10 primers could be optimized. Three primer pairs performed best at Ta of 54°C. The optimum concentration of MgCl2 was found to be 1.5mM for all primer pairs. Similarly the number of PCR cycles was found to be 40 for all 10 primer pairs except for primer pair 57. Optimized PCR conditions were used for subsequent studies such as transferability of EST primers to other Fagopyrum species and construction of linkage map.Nepal Agric. Res. J. Vol. 8, 2007, pp. 1-6DOI: http://dx.doi.org/10.3126/narj.v8i0.11563

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Xu, Eugenia Y., Lisa M. Schneper, and Daniel A. Notterman. "A novel metric to improve mismatched primer selection and quantification accuracy in amplifying DNA repeats for quantitative polymerase chain reactions." PLOS ONE 18, no. 10 (October 9, 2023): e0292559. http://dx.doi.org/10.1371/journal.pone.0292559.

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In quantitative polymerase chain reaction (qPCR) experiments, primers containing mismatches with respect to the template are widely used in measuring repetitive DNA elements. Primer-template mismatches may lead to underestimation of the input sample quantity due to inefficient annealing and amplification. But how primer-template mismatches affect quantification accuracy has not been rigorously investigated. In this study, we performed a series of qPCR experiments in which we tested three pairs of mismatched telomere primers (tel1/tel2, tel1b/tel2b and telg/telc) and two pairs of perfect-match reference gene primers (36B4-F/-R and IFNB1-F/-R) at three different primer concentrations under four cycling conditions. Templates used were genomic DNA from two human cell lines and oligo duplexes which contained telomere sequences, reference gene sequences, or both. We demonstrated that the underestimation of input sample quantity from reactions containing mismatched primers was not due to lower amplification efficiency (E), but due to ineffective usage of the input sample. We defined a novel concept of amplification efficacy (f) which quantifies the effectiveness of input sample amplification by primers. We have modified the conventional qPCR kinetic formula to include f, which corrects the effects of primer mismatches. We demonstrated that reactions containing mismatched telomere primer pairs had similar efficiency (E), but varying degrees of reduced efficacy (f) in comparison to those with the perfect-match gene primer pairs. Using the quantitative parameter f, underestimation of initial target by telomere primers can be adjusted to provide a more accurate measurement. Additionally, we found that the tel1b/tel2b primer set at concentration of 500 nM and 900 nM exhibited the best amplification efficacy f. This study provides a novel way to incorporate an evaluation of amplification efficacy into qPCR analysis. In turn, it improves mismatched primer selection and quantification accuracy in amplifying DNA repeats using qPCR methods.

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Matos-Oliveira, Carla Faine, Cibelle Santos Dias, Elisa Susilene Lisboa Santos, and Carlos Bernard Moreno Cerqueira Silva. "Caracterização e seleção de marcadores ISSR para análise genético-populacional de Atta sexdens (Hymenoptera: Formicidae)." Multi-Science Journal 1, no. 9 (March 21, 2018): 4. http://dx.doi.org/10.33837/msj.v1i9.353.

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Com o intuito de gerar subsídios para realização de estudos genéticos em formiga cortadeira (especificamente Atta sexdens), caracterizou-se o padrão de amplificação de DNA gerado por primers ISSR. Para tanto, operárias médias de A. sexdens tiveram DNA genômico extraído utilizando a cabeça, mesossoma e pernas. Os padrões de amplificação foram analisados em géis de agarose 2%. Dentre os 23 primers ISSR testados 17 geraram produtos de amplificação (sendo 12 destes primers classificados como “tipo ideal” e cinco como “tipo razoável”). O percentual de polimorfismo variou entre 20% (primer TriAGA3’RC) e 100% (primer DiCA3’YG). Os resultados atestam que os primers ISSR selecionados apresentam um grau de polimorfismo considerável e, portanto, são adequados para estudos de diversidade genética em formigas cortadeiras, especialmente para A. sexdens.

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Kilpatrick, David R., Baldev Nottay, Chen-Fu Yang, Su-Ju Yang, Edson Da Silva, Silvia Peñaranda, Mark Pallansch, and Olen Kew. "Serotype-Specific Identification of Polioviruses by PCR Using Primers Containing Mixed-Base or Deoxyinosine Residues at Positions of Codon Degeneracy." Journal of Clinical Microbiology 36, no. 2 (1998): 352–57. http://dx.doi.org/10.1128/jcm.36.2.352-357.1998.

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We have developed a method for determining the serotypes of poliovirus isolates by PCR. Three sets of serotype-specific antisense PCR-initiating primers (primers seroPV1A, seroPV2A, and seroPV3A) were designed to pair with codons of VP1 amino acid sequences that are conserved within but that differ across serotypes. The sense polarity primers (primers seroPV1S, seroPV2S, and seroPV3S) matched codons of more conserved capsid sequences. The primers contain mixed-base and deoxyinosine residues to compensate for the high rate of degeneracy of the targeted codons. The serotypes of all polioviruses tested (48 vaccine-related isolates and 110 diverse wild isolates) were correctly identified by PCR with the serotype-specific primers. None of the genomic sequences of 49 nonpolio enterovirus reference strains were amplified under equivalent reaction conditions with any of the three primer sets. These primers are useful for the rapid screening of poliovirus isolates and for determining the compositions of cultures containing mixtures of poliovirus serotypes.

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BĂDULESCU, Adriana, Carmen F. POPESCU, Anamaria M. DUMITRU, and Dorin I. SUMEDREA. "New varieties of tomato - morphological aspects and molecular characterisation with RAPD and SSR markers." Notulae Scientia Biologicae 12, no. 4 (December 21, 2020): 818–28. http://dx.doi.org/10.15835/nsb12410841.

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This study presents the main morphological features and the first molecular investigations of four new tomato varieties (Solanum lycopersicum), aiming to obtain their complete characterisation. Evaluation with the standard descriptors for tomato revealed specific and distinct traits for each analysed variety. The molecular analyses for variety identification started with testing three methods for DNA extraction. With an optimized method, which doesn’t need liquid nitrogen for plant tissue disruption, good quality DNA was obtained, in adequate quantities, and well preserved when stored at -20 °C. To highlight the genetic differences among the analysed tomato varieties, nine RAPD primers and ten SSR primers were tested. Of these, the optimal amplification protocols for five RAPD primers and five SSR primers were established. The amplified products obtained with RAPD primers revealed an average number of bands per primer of 8.8 and a total rate of polymorphism of 59.1%; with OPB10 primer was seen the highest number of DNA bands (11), and with OPA07 primer was registered the highest degree of genetic variability among the studied varieties (77.7%). Two SSR markers (SSR 20 and SSR T107) amplified monomorphic banding patterns corresponding to 170 base pairs and 250 base pairs, respectively, for all varieties; with SSR T7, SSR T62, and SSR T70 primers were generated multiple amplification bands, with a different distribution of the bands into the agarose gel for each analysed tomato variety.

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JAYARAO, B. M., B. E. GILLESPIE, and S. P. OLIVER. "Application of Randomly Amplified Polymorphic DNA Fingerprinting for Species Identification of Bacteria Isolated from Bovine Milk." Journal of Food Protection 59, no. 6 (June 1, 1996): 615–20. http://dx.doi.org/10.4315/0362-028x-59.6.615.

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A polymerase chain reaction-based DNA fingerprinting system for species identification of bacteria in milk was developed using randomly amplified polymorphic DNA. A total of 108 organisms including 24 ATCC reference strains and 84 wild-type isolates belonging to gram-negative, Staphylococcus, Enterococcus, and Streptococcus species were used to develop the system. Organisms included in the study were those that are isolated frequently from milk. Forty primers from two commercially available primer kits were evaluated to determine the “ideal” primer that could be used for several bacterial species. Over 960 DNA fingerprint patterns were analyzed by laser densitometry. Seven of the 40 primers met criteria established for primer selection. However, only primers OPE-4 (5′ GTGACATGCC-3′) and OPE-20 (5′-AACGGTGACC-3′) allowed differentiation between all 19 ATCC bacterial species included in the study. The other five primers were restricted to either gram-negative bacteria (OPA-7, OPA-14), Staphylococcus species (OPA-13, OPA-14, OPA-18), or Streptococcus species (OPA-3). Primers OPE-4 and OPE-20 were further evaluated using 84 wild-type isolates. A bacterial species identification scheme was developed based on characteristic polymorphic DNA fragments obtained with primers OPE-4 and OPE-20. Results of this study suggest that RAPD fingerprinting has the potential for being developed into a rapid and accurate method for species identification of bacteria in milk.

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Latvis, Maribeth, Sebastian M. E. Mortimer, Diego F. Morales-Briones, Samuel Torpey, Simon Uribe-Convers, Sarah J. Jacobs, Sarah Mathews, and David C. Tank. "Primers for Castilleja and their Utility Across Orobanchaceae: I. Chloroplast Primers." Applications in Plant Sciences 5, no. 9 (September 2017): 1700020. http://dx.doi.org/10.3732/apps.1700020.

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YE, Hui, Yun-Long LIU, Bing-Jie ZOU, Hai-Ping WU, and Guo-Hua ZHOU. "Design of Polymerase Chain Reaction Primers and Sequencing Primers in Pyrosequencing." CHINESE JOURNAL OF ANALYTICAL CHEMISTRY (CHINESE VERSION) 41, no. 5 (2013): 744. http://dx.doi.org/10.3724/sp.j.1096.2013.20924.

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Utami, Ni Made Ayuratih, I. Gede Putu Wirawan, and I. Ketut Suada. "PRIMER DESIGN OF CVPDr DNA FRAGMENT SEQUENCES THAT AMPLIFY SPECIFIC FRAGMENTS TO DISTINCT THE RESISTANT FRAGMENT FROM Triphasia trifolia (Burm. F.) P. Wils. AND THE SUSCEPTIBLE FRAGMENT FROM Citrus nobilis Lour." International Journal of Biosciences and Biotechnology 7, no. 2 (June 16, 2020): 91. http://dx.doi.org/10.24843/ijbb.2020.v07.i02.p05.

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CVPDr is a DNA fragment that indicates that plants are resistant to CVPD. Previous research using primers that amplified 841 bp CVPDr fragment was able to amplify the fragment from Triphasia trifolia that considers being a resistant plant, Citrus aurantifolia var. seedless which considers being a tolerant plant, and some susceptible citrus plants to CVPD disease. In this study, we designed some primers that amplified only CVPDr DNA fragment from T. trifolia which consider as the resistant plant and a primer that amplified only DNA fragmen from Citrus nobilis which consider as the susceptible citrus plants. The primers for CVPDr on T. trifolia are TCATCTGCATGGGATACC for forward primer and GCCTTGAGCTTGTAAGTG for reverse primer which turned out to amplify the DNA of T. trifolia and also the C. nobilis cultivar Denpasar and only succeeded in not amplifying the C. nobilis cultivar Gianyar. The primers for CVPDr on C. nobilis are GAATGGCTTAGCAGAAAGG for forward primer and GGTTGTAGATGGACATAGG for reverse primer turned out can not only amplify the DNA C. nobilis but also amplify T. trifolia.

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Journal articles on the topic 'Primers'

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