Search

Lychnophora pinaster Mart. (Asteraceae) is a Brazilian medicinal plant, extensively employed in popular medicine as an anti-inflammatory, analgesic and healing agent. Thirteen polymorphic microsatellite markers were developed and optimized for L. pinaster from an enriched genomic library. The markers were used to analyse 37 plants from two native populations, generating an average number of 6.6 alleles per polymorphic locus. These loci are important tools for future studies of population genetics. [ABSTRACT FROM AUTHOR]

• The detection efficiency of ten (10) of the most utilized and novel CSSV primers were evaluated on isolates of the virus across Ghana through PCR assays. • Results from the PCR assays showed a highly variable and poor efficiency of the primers on the 189 samples of CSSV isolates evaluated. • Primer detection efficiency ranged from 4 % to 23 % with the four best performing primers being P4 (23%) > CSSD1 (21 %) > CSSD2 and BADNA (19 %). • The generally poor and inconsistent efficiency of the primers are discussed in the context of the genetic variability and also the occurrence of new variants of the virus in Ghana. Reliable diagnostic tools capable of detecting latent and asymptomatic infections are critically important to support the management of the cocoa swollen shoot virus disease (CSSVD) and also to complement research activities on screening for resistant cocoa varieties. Development of efficient polymerase chain reaction (PCR) assays sensitive for detection of CSSV infections has thus been a major research focus over the years. Advances in the full genome sequence information have resulted in the design of several Cacao swollen shoot virus (CSSV)-specific and degenerate primers. The objective of the present study was to further assess the detection efficiency of ten (10) of the most utilized and novel CSSV primers on isolates of the virus across Ghana. Results from the PCR assays showed a highly variable and poor efficiency of the primers on the 189 samples of CSSV isolates evaluated. The overall detection potential of the primers ranged from 4 % to 23 % with the four best performing primers in terms of PCR positivity being P4 (23 %), CSSD1 (21 %), JOEL (21 %) CSSD2 (19 %) and BADNA (19 %). The generally poor and inconsistent efficiency of the primers are discussed in the context of the genetic variability and also the occurrence of new variants of the virus in Ghana. [ABSTRACT FROM AUTHOR]

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

The tropical climate present in the Yucatan Peninsula is one of the most aggressive environments found in Mexico, which results in many concrete structures being affected by corrosion. Several types of primers. which are locally available, are used as prevention and/or repair methods for reinforcement steel found in concrete structures. Nonetheless, such primers are not designed to be used under alkaline environmental conditions. The objective of this investigation was to evaluate the efficiency of these primers used to repair concrete columns of buildings exposed to a coastal marine environment in southeast Mexico. The repair method used involved the following steps: removal of the old concrete, cleaning the rebar with a brush, applying primer to the reinforcement steel, arid finally applying bonding agent and new concrete cover. The electro-chemical behavior of repaired and adjacent (non-repaired) zones was monitored based on electrochemical techniques with the use of a commercially confined-current corrosimeter. Variables measured were polarization resistance, corrosion potential, and concrete electrical resistivity. One reference column was used for which primer application was not conducted, and four other repair systems were used for which different permeable primers were tested. Theoretically, the primers used should provide protection mechanisms against corrosion, such as repassivation, inhibition, barrier, and cathodic protection. Results showed that the barrier system worked best, followed by the cathodic protection and inhibitor mechanisms. Overall, these findings suggest that primer quality was the most relevant parameter. Primer application not only decreased the corrosion rate of repaired zones, but also the galvanic couple, which apparently only lasted a few days. Primers tested here proved to be a reasonable, low-cost option for localized repairs in cases where the concrete is of inferior quality. Nonetheless, long-term performance of repaired structures must be evaluated through careful monitoring. [ABSTRACT FROM AUTHOR]

We describe primers and polymerase chain reaction conditions to amplify 21 tetranucleotide microsatellite DNA loci in black bears ( Ursus americanus). We tested primers using individuals from two populations, one each in Georgia and Florida. Among individuals from Georgia ( n = 29), primer pairs yielded an average of 2.9 alleles (range, one to four) and an average observed heterozygosity ( HO) of 0.50 (range, 0.00 to 0.79). Among individuals from Florida ( n = 19), primer pairs yielded an average of 5.7 alleles (range, one to 14) and an HO of 0.55 (range, 0.00 to 1.00). A comparison of previously developed markers with individuals from Georgia suggests that bear populations in Georgia and Florida have reduced allelic diversity relative to other populations. [ABSTRACT FROM AUTHOR]

We characterized 24 polymorphic tetranucleotide microsatellite loci for delta smelt ( Hypomesus transpacificus) endemic to the San Francisco Bay Estuary, CA, USA. Screening of samples ( n = 30) yielded two to 26 alleles per locus with observed levels of heterozygosity ranging from 0.17 to 1.0. Only one locus deviated from Hardy–Weinberg equilibrium, suggesting these individuals originate from a single panmictic population. Linkage disequilibrium was found in two pairs of loci after excluding the locus out of Hardy–Weinberg equilibrium. Twenty-two primer pairs cross-amplified in wakasagi smelt ( Hypomesus nipponensis), and 15 primer pairs cross-amplified in longfin smelt ( Spirinchus thaleichthys). [ABSTRACT FROM AUTHOR]

Aerolysin is a hemolytic toxin encoded by aerolysin gene (1482 bp) that plays a key role in the pathogenesis of Aeromonas hydrophila infection in fish. New speciesspecific primers were designed to amplify 326 bp conserved region of aerolysin gene for A. hydrophila. Twenty-five isolates of A. hydrophila recovered from fish and pond water were studied for detection of aerolysin gene. Aerolysin gene was detected in 85% of the isolates during the study. The designed primers were highly specific and showed no cross reactivity with Escherichia coli, Aeromonas veronii, Vibrio cholerae, Flavobacterium spp., Chyseobacterium spp. and Staphylococcus aureus. The sensitivity limit of primers for detection of aerolysin gene in the genomic DNA of A. hydrophila was 5 pg. [ABSTRACT FROM AUTHOR]

Microsatellite loci were isolated for Ceratosolen solmsi, pollinator of the dioecious Ficus hispida. We developed nine polymorphic microsatellite loci based on the method of polymerase chain reaction isolation of microsatellite arrays (PIMA). Enrichment of genomic libraries was performed by random amplified polymorphic DNA (RAPD). A subset of 38 positive clones was sequenced; 15 clones showed microsatellite loci. We tested 15 designed primer pairs and nine of them produced polymorphic amplification in 48 individual wasps collected from different fruits of the dioecious host fig Ficus hispida in China. Among the 48 individuals, 49 alleles were obtained at the nine loci. The observed heterozygosity ranged between 0.357 and 0.634. [ABSTRACT FROM AUTHOR]

Abstract: Studies of the human cytomegalovirus (HCMV) glycoprotein N (gpUL73-gN) showed that genotypic variations exist in different geographic areas, with gN-2 unidentified in Chinese population. The purpose of this study was to determine the HCMV gN variants in the Chinese population of Taiwan. Primers were designed and a polymerase chain reaction (PCR) was carried out on the UL73 gene. The PCR products were subjected to restriction fragment length polymorphism (RFLP) analysis. The same PCR-RFLP assay was repeated using primers published previously to demonstrate the influence of primer design. Of the 48 clinical HCMV isolates, 33 were positive for PCR products by both primer sets. Fifteen were positive only by the “in-house” PCR. The distribution of gN-1, gN-2, gN-3, and gN-4 by RFLP analysis was 14:11:7:17, with one isolate positive for both gN-1 and gN-2. The published primers detected the four genotypes with the number of 14:0:2:17. The under-representation of gN-2 and gN-3 by the method published previously may be due to inappropriate primer design when re-examining the sequences. On the basis of the results of this study, gN-2 is not the rarest gN genotype in the Chinese population of Taiwan. The design of primers used for PCR-RFLP genotyping may have a great influence on the frequency distribution of HCMV genomic variants. [Copyright &y& Elsevier]

One hundred and thirty-five microsatellite markers were developed for hop Humulus lupulus L. from di- and trinucleotide-enriched libraries. Seventy-eight primers showed amplification in two tested genotypes. Twenty-four loci were further characterized on a population of 34 hop samples and the number of alleles per locus, observed heterozygosity and expected heterozygosity ranged from two to 20 (9.7 on average), from 0.0294 to 0.9412 (0.6234 on average) and from 0.0294 to 0.9170 (0.6720 on average), respectively. These microsatellite markers will be further used for studying population structures and relationships and for identifying important qualitative and quantitative loci of hop. [ABSTRACT FROM AUTHOR]

A unique DNA isolation protocol based on microwave irradiation and density centrifugation for the simultaneous detection of porcine, chicken, and mutton meat species in food by PCR using newly developed primers is described. Emphasis is placed on the detection of pork, as it is considered unlawful in the diet in major religions such as Islam and Judaism. The method has been optimized using the primers designed from porcine mitochondrial DNA (mtDNA) sequence and tested in 96 samples. The method involves 35 seconds of high microwave irradiation of lysed sample homogenate followed by a 5- min centrifugation in 1.5 mL Eppendorf tubes until a clear supernatant is obtained. The design of this protocol makes it possible to process many samples in a short time. To evaluate the method pork and food spiked with different concentrations of pork together with unknown food samples were analyzed by PCR using the new set of porcine specific primers. The new set of primers and the method involved showed high sensitivity in detecting porcine and chicken content in food in addition as a marker for the detection of mutton. The present sample preparation method has the potential to be applied to other meat detection systems as well. [ABSTRACT FROM AUTHOR]

Abstract: Recently, as genome-scale data have become available for more organisms, the development of phylogenetic markers from nuclear protein-coding loci (NPCL) has become more tractable. However, new methods are needed to efficiently sort the large number of genes from genomic databases into more limited sets appropriate for particular phylogenetic questions, while avoiding introns and paralogs. Here we describe a general methodology for identifying candidate single-copy NPCL from genomic databases. Our method uses information from reference genomes to identify genes with relatively large continuous protein-coding regions (i.e., ⩾700bp). BLAST comparisons are used to help avoid genes with paralogous copies or close relatives (i.e., gene families) that might confound phylogenetic analyses. Exon boundary information is used to identify appropriately spaced potential priming sites. Using this method, we have developed over 25 novel NPCL, which span a variety of desirable evolutionary rates for phylogenetic analyses. Although targeted for higher-level phylogenetics of squamate reptiles, many of these loci appear to be useful across and within other vertebrate clades (e.g., amphibians), and some are relatively rapidly evolving and may be useful for closely-related species (e.g., within genera). This general method can be used whenever large-scale genomic data are available for an appropriate reference species (not necessarily within the focal clade). The method is also well suited for the development of intron regions for lower-level phylogenetic and phylogeographic studies. We provide an online database of alignments and suggested primers for approximately 85 NPCL that should be useful across vertebrates. [Copyright &y& Elsevier]

Increasing the number of characters used in phylogenetic studies is the next crucial step towards generating robust and stable phylogenetic hypotheses - i.e., strongly supported and consistent across reconstruction method. Here we describe a genomic approach to finding new protein-coding genes for systematics in nonmodel taxa, which can be PCR amplified from standard, slightly degraded genomic DNA extracts. We test this approach on Lepidoptera, searching the draft genomic sequence of the silk moth Bombyx mori, for exons > 500 bp in length, removing annotated gene families, and compared remaining exons with butterfly EST databases to identify conserved regions for primer design. These primers were tested on a set of 65 taxa primarily in the butterfly family Nymphalidae. We were able to identify and amplify six previously unused gene regions (Arginine Kinase, GAPDH, IDH, MDH, RpS2, and RpS5) and two rarely used gene regions (CAD and DDC) that when added to the three traditional gene regions (COI, EF-1α and wingless) gave a data set of 8114 bp. Phylogenetic robustness and stability increased with increasing numbers of genes. Smaller taxanomic subsets were also robust when using the full gene data set. The full 11-gene data set was robust and stable across reconstruction methods, recovering the major lineages and strongly supporting relationships within them. Our methods and insights should be applicable to taxonomic groups having a single genomic reference species and several EST databases from taxa that diverged less than 100 million years ago. [ABSTRACT FROM AUTHOR]

Abstract: This report presents PRImer Selector (PRISE), a new software package that implements several features that improve and streamline the design of sequence-selective PCR primers. The PRISE design process involves two main steps. In the first step, target and non-target DNA sequences are identified. In the second step, primers are designed to amplify target (but not non-target) sequences. One important feature of PRISE is that it automates the task of placing primer–template mismatches at the 3'' end of the primers — a property that is crucial for sequence selectivity. Once a list of candidate primers has been produced, sorting tools in PRISE speed up the selection process by allowing a user to sort the primers by properties such as amplicon length, GC content and sequence selectivity. PRISE can be used to design primers with a range of specificities, targeting individual sequences as well as diverse assemblages of genes. PRISE also allows user-defined primers to be analyzed, enabling their properties to be examined in relation to target and non-target sequences. The utility of PRISE was demonstrated by using it to design sequence-selective PCR primers for an rRNA gene from the fungus Pochonia chlamydosporia. [Copyright &y& Elsevier]

Pour alphabétiser et instruire tous les enfants, la plupart des nations occidentales ont rendu l'école obligatoire à la fin du XIXe siècle. Alphabétisation, éducation universelle de base et scolarisation obligatoire sont ainsi devenues trois réalités indissociables pour les habitants de ces pays et un modèle de référence pour le reste du monde. Aujourd'hui, l'écriture est l'outil sans lequel aucun développement économique, politique, culturel n'est imaginable. «Literacy for all is at the heart of basic education for all» and «creating literate environments and societies is essential for achieving the goals of eradicating poverty, reducing child mortality, curbing population growth, achieving gender equality and ensuring sustainable development, peace and democracy», dit le rapport de l'UNESCO, paru en avril 2006, intitulé Literacy for Life... [ABSTRACT FROM AUTHOR]

The article focuses on the selection of DNA markers and primers. The authors begin by describing an algorithm designed to detect substrings that have edit distances to a fixed substring at most equal to a given error, followed by proposing an algorithm which finds the set of all substrings that have edit distances larger than errors to all others. Then, the authors provide their practical applications and implications, focusing on biological issues such as hairpins and guanine-cytosine percentage. They conclude that primers and DNA markers, which have high chance of performing well in experiments and microarrays, can be selected.

Thirteen polymorphic microsatellites were isolated from Leuciscus cephalus, a widespread cyprinid species with great ecological tolerance. Together with the cross-species amplification of six additional loci originally published for three cyprinid fish species, we optimized a multiplex panel for L. cephalus allowing the genotyping of 19 polymorphic loci. Number of alleles and heterozygosity per locus in a sample of 20 fish individuals ranged from two to 16 and from 0.05 to 0.90, respectively. These primers will be useful in determining the population structure of L. cephalus. In addition, successful cross-amplification was obtained for several species of Cyprinidae. [ABSTRACT FROM AUTHOR]

We present a new set of 15 polymorphic microsatellite primer sequences developed from Ficus carica L. The variability of specific microsatellite regions was assessed in wild population of figs from the northern Adriatic coast and all 15 primer pairs showed single-locus amplification with a total of 65 alleles and an observed heterozygosity ranging from 0.285 to 0.863. The 15 new microsatellite loci represent a significant tool for population genetic structure studies and will be further used to investigate the origin and maintenance of genetic variation within and between populations of figs along the Adriatic coastal region. [ABSTRACT FROM AUTHOR]

The measurement of vitellogenin (vtg) gene transcription has been shown to be a reliable indicator of exposure to estrogenic compounds. Unfortunately, the relatively poor molecular characterization of North American fish species has hindered its application to a larger number of ecologically important species. The current research aimed to demonstrate specific amplification of vtg gene transcripts in three model (zebrafish, rainbow trout, and medaka) and six nonmodel (emerald shiner, pearl dace, smallmouth bass, creek chub, white sucker, and golden redhorse) fish species. Quantitative polymerase chain reaction (QPCR) primers for model species were designed from publicly available vtg sequences. Successful amplification of vtg was demonstrated in fish exposed to 17α-ethinylestradiol (EE2) for all model species. Vitellogenin primers for selected nonmodel species were designed from published sequences of closely related species. Multiple primers were developed targeting different regions of the vtg gene. The successful amplification of vtg was confirmed through size and sequence analysis for all nonmodel species with the exception of the white sucker, in which amplifications failed. Furthermore, QPCR primers and conditions were quantitative over five orders of magnitude in at least one species (pearl dace) exposed to 5 ng/L of EE2 for 24 h. The selected species are found in a wide array of ecological habitats that span the United States. Inclusion of vtg transcriptional analysis for wild, ecologically relevant fish in monitoring studies may aid in understanding the extent of estrogenic exposure in aquatic ecosystems across the United States. [ABSTRACT FROM AUTHOR]

RAPD (random amplified polymorphic DNA) and ISSR (inter simple sequence repeats) markers were used to study DNA polymorphism of 48 assorted accessions (plus trees) of Tectona grandis at the National Teak Germplasm Bank, Chandrapur, India. In general, the germplasm exhibited a very high level of molecular diversity and DNA polymorphism. Molecular diversity based on Jaccard's similarity coefficients among 48 plus trees ranged from 0.31-0.85 in RAPD assay and 0.27-0.88 in ISSR assay. RAPD primers amplified more loci than ISSR primers. However, RAPD and ISSR primers amplified 93.2 and 95.9% polymorphic loci respectively. Jaccard's similarity coefficient matrices for both molecular marker systems exhibited a poor correlation (r = 0.36) among themselves and clustering pattern of plus trees did not strictly follow their territorial distribution, possibly due to frequent movement of seeds/planting stocks across localities/regions. From application point of view, RAPD primers amplified several loci specific to 16 plus trees while ISSR primers, few loci specific to three plus trees. These loci can be sequenced and developed into SCAR (sequence characterized amplification region) markers for genotype fingerprinting or development of specific DNA probes for identification of clones or ramets of teak. [ABSTRACT FROM AUTHOR]

We describe primers and polymerase chain reaction conditions to amplify 15 tetranucleotide microsatellite loci from the loggerhead sea turtle ( Caretta caretta). The primers were tested on 30 individuals that nested along the Georgia, USA coast. The primer pairs developed in this study yielded an average of 13.9 alleles per locus (range of 10–21), an average observed heterozygosity of 0.91 (range 0.79–1.00), and an average polymorphic information content of 0.88 (range 0.84–0.92). [ABSTRACT FROM AUTHOR]

Abstract: We report the first cases of locally-acquired Diphyllobothrium nihonkaiense (Yamane, Kamo, Bylund and Wikgren, 1986) in Switzerland, confirmed by genetic analysis (18S rRNA, COI and ITS1-5.8S rRNA-ITS2 genes). Diphyllobothriasis in this country is attributed to the tapeworm D. latum (Linnaeus, 1758) but the increasing popularity of raw fish culinary specialities (sushi, carpaccio, tartare) brings out a new diagnostic problem, so that people can get infected by exotic species of tapeworms. [Copyright &y& Elsevier]

Costus speciosus, an important medicinal plant species found in the Andaman and Nicobar Islands, was collected from 14 localities through recurrent survey. RAPD-PCR analysis involving 12 decamer random primers was used to assesses the quantum of genetic variation at genomic level. Four primers showed appreciable intra-species variation or molecular polymorphism at amplicon levels. Despite morphological identity, a great deal of polymorphism was observed among the accessions. UPGMA analysis showed ~35% variation in the collections, which is deemed to be useful in formulating sound conservation strategies for this precious medicinal plant species under the humid tropics of Bay Islands. [ABSTRACT FROM AUTHOR]

The cacao plant, Theobroma cacao L., produces white seeds (beans) that form the major ingredient of processed chocolate. A great deal of research effort has been expended to the development of new genetically modified cacao plants with improved productivity and resistance and beans of good industrial quality. The availability of suitable genetic markers is an important aspect of the efficient selection and breeding of this perennial species. We describe the development of 123 microsatellite loci of cacao. An optimized protocol was used to construct and screen a microsatellite-enriched genomic library from which we isolated 64 di-nucleotide, 45 tri-nucleotide and 14 tetra-nucleotide microsatellite loci. The primers were tested on samples from five different T. cacao accessions, one accession from T. grandiflorum and one accession from Herranea sp. Among the 123 loci, 54 were polymorphic, 61 were monomorphic and eight did not present an amplification product. These new markers will be useful in future studies by increasing the accuracy of genotypic assessments in diverse cocoa tree populations as well as in other species of the Theobroma genus. [ABSTRACT FROM AUTHOR]

Fourteen polymorphic microsatellites were isolated from Neogobius kessleri, a benthic fish of Ponto-Caspian origin which has been recently introduced into the Middle and Upper Danube River. Number of alleles and heterozygosity per locus in a sample of 32 fish individuals ranged from two to four and from 0.13 to 0.75, respectively. These primers will be useful in determining the population structure of N. kessleri. In addition, successful cross-amplification was obtained for four related species, N. melanostomus, N. fluviatilis, N. gymnotrachelus and Proterorhinus marmoratus. These microsatellite loci may be useful for the evaluation of the origin of non-native goby populations. [ABSTRACT FROM AUTHOR]

ObjectivesTo characterize the effect of two thione metal primers with phosphate groups on the surface morphology and composition of two noble prosthodontic alloys.MethodsCast specimens from Argen 81(Au–Pd) and Argipal (Hi–Pd) alloys which were ground, polished and ultrasonicated in water, were divided in two groups (2×3) and treated with single layers of Alloy Primer (AP) and Metal Primer II (MP) primers respectively. The treated alloy surfaces were washed off with acetone and then examined by polarized light microscopy (PLM), reflection FTIR microspectroscopy (FTIRM) and X-ray photoelectron spectroscopy (XPS).ResultsAfter AP treatment, PLM revealed a crystalline phase (VBATDT) dispersed in an amorphous phase (MDP plus soluble VBATDT) on both the alloys tested. MP demonstrated a fibrial arrangement with the most dense structure found on the Hi–Pd alloy. FTIRM failed to clearly resolve the presence of SH peaks on alloy surfaces. Moreover, NH and PS peaks were identified denoting the presence of original thione tautomers. In both primers, phosphates were detected in a dissociative state (-PO32−). FTIR molecular mapping confirmed separation of VBATDT from MDP and MEPS from residual MMA. XPS showed that on alloy surfaces approximately 50% of sulphur was in the sulphide state, the rest being organic sulphur. AP showed higher sulphide percentage than MP on both alloys and higher sulphide percentage on the Au–Pd alloy (p < 0.05).Clinical significancePhase separation of the primer components on alloy surfaces may adversely affect their clinical performance. Sulphide formation on alloy surfaces was confirmed only by XPS under ultra-high vacuum and not by environmental techniques like FTIR; this poses serious questions on the chemical bonding capacity of these primers with the noble alloys tested under environmental conditions. [Copyright &y& Elsevier]

Relative quantification real-time PCR was used to quantify several bacterial species in ruminal samples from two lactating cows, each sampled 3 h after feeding on two successive days. Abundance of each target taxon was calculated as a fraction of the total 16S rRNA gene copies in the samples, using taxon-specific and eubacterial domain-level primers. Bacterial populations showed a clear predominance of members of the genus Prevotella, which comprised 42% to 60% of the bacterial rRNA gene copies in the samples. However, only 2% to 4% of the bacterial rRNA gene copies were represented by the classical ruminal Prevotella species Prevotella bryantii, Prevotella ruminicola and Prevotella brevis. The proportion of rRNA gene copies attributable to Fibrobacter succinogenes, Ruminococcus flavefaciens, Selenomonas ruminantium and Succinivibrio dextrinosolvens were each generally in the 0.5% to 1% range. Proportions for Ruminobacter amylophilus and Eubacterium ruminantium were lower (0.1% to 0.2%), while Butyrivibrio fibrisolvens, Streptococcus bovis, Ruminococcus albus and Megasphaera elsdenii were even less abundant, each comprising <0.03% of the bacterial rRNA gene copies. The data suggest that the aggregate abundance of the most intensively studied ruminal bacterial species is relatively low and that a large fraction of the uncultured population represents a single bacterial genus. [ABSTRACT FROM AUTHOR]

Summary: The saline pond microalga, Dunaliella salina (Dunal) Teod. maintained in De Walne''s (basal) medium under laboratory conditions was confirmed by amplifying the chromosomal DNA of the microalga by PCR with specific primers MA1 and MA2. Seaweed extracts obtained from Sargassum wightii and Ulva lactuca were amended separately at 1.0%, 1.5%, 2.0% and 2.5% levels to the basal medium in order to assess their potential on the growth and concentration of pigments, viz. Chl a, Chl b and β-carotene of the alga. β-Carotene was isolated and visible absorption spectrum was taken at 443 and 475nm confirmed the presence of 9-cis-β-carotene and all-trans-β-carotene isomers. Maximum yield, highest division rate (μ) and highest pigment concentrations were observed in the cells grown in 1.5% S. wightii and 2.0% U. lactuca amended medium and these cells were subjected to DAPI staining. The results of epifluorescence microscopy and image analysis revealed a significant enhancement of the cell and nuclear area of the microalgae. [Copyright &y& Elsevier]

Thirty-three microsatellite loci were isolated for the Australian rainforest tree Macadamia integrifolia. Genotyping across a test panel of 43 commercial cultivars generated an average polymorphic information content of 0.480. Five loci showed no polymorphism across cultivars. Significant linkage disequilibrium was detected in 10 pairwise comparisons, including two pairs of loci identified from the same clone sequence. The 33 microsatellite loci represent a significant tool for genome mapping and population genetic studies. [ABSTRACT FROM AUTHOR]

We describe primers and polymerase chain reaction (PCR) conditions to amplify 18 tetranucleotide microsatellite DNA loci in eastern bluebirds ( Sialia sialis). The primers were tested using individuals from two study sites in Georgia and South Carolina. Among individuals from the Georgia population ( n = 23), the primer pairs developed in this study yielded an average of 6.6 alleles per locus (range 2–12), an average observed heterozygosity of 0.56 (range 0.24–0.96) and an average polymorphic information content of 0.65 (range 0.3–0.86). Among individuals from the South Carolina population ( n = 19), the primer pairs yielded an average of 5.8 alleles per locus (range 2–9), an average observed heterozygosity of 0.56 (range 0.05–0.86) and an average polymorphic information content of 0.63 (range 0.29–0.83). [ABSTRACT FROM AUTHOR]

Genomic DNA assays of bee races Apis mellifera caucasica and Apis mellifera carnica and three lines of the latter were performed with eleven simple sequence repeat primers. Using these primers in PCR there were amplified 60 DNA fragments of which 66.7% were found to be polymorphic. The number of fragments produced in the DNA profiles of different bee races and lines varied from 2 to 10, with their sizes varying within 350-3000 bp. The genetic diversity of the individuals of A. m. caucasica and A. m. carnica races was revealed by using AC(GACA)4, (GACA)4GT, (ATG)5GA, (TCC)5GT, (AC)8YT and (CT)5A primers in PCR. Race and line-specific DNA profiles were obtained. Fragments specific to A. m. caucasica race were produced using (TCC)5GT, (AC)8YT and (AC)8T primers in PCR, and those specific to A. m. carnica lines were amplified by (AC)8YT primer. Having used eleven simple sequence repeat primers for DNA fingerprinting of bees, on average in 6.8% of the cases there were generated 5-7 types of DNA profiles, in 70.4% of cases 2-4 types of profiles, in the rest of 22.8% of cases any no polymorphism was revealed between the bee races and within the lines tested. [ABSTRACT FROM AUTHOR]

Abstract: Leaching profiles for two chromate-inhibited epoxy polyamide primers have been analysed and non-Fickian diffusion kinetics have been observed. The time dependence was found to be t 0.25 instead of the standard Fickian t 0.5. The t 0.25 time dependence was consistent for different pH values (1, 3, 5 and 7) even though the overall release rates increased with decreasing pH. Abrading the primer surface increased the overall leaching rate but did not change the t 0.25 dependence. The unusual release kinetics cannot be explained by a decreasing effective diffusion coefficient (via changes in porosity and tortuosity or a fractal structure) or pH-dependent chemical reactions. The leaching rates have been modelled using a simple power law, a polynomial, and the shrinking core model. The polynomial model produced the best fit to leaching data and provides some insight into the source of the non-Fickian leaching behaviour. [Copyright &y& Elsevier]

Abstract: Leaching and characterisation studies have been undertaken on two chromate-inhibited epoxy polyamide primers. Leaching was carried out in 5% (w/v) NaCl solutions at different pH values (1, 3, 5 and 7) and the amount of Cr released into solution was monitored over time. Cr release was initially high, but as the immersion time increased the leaching from the primers slowed. Prior to and after immersion, the primers were characterised by a number of techniques including electron microprobe analysis, X-ray microdiffraction, Raman spectroscopy, and positron annihilation lifetime spectroscopy. The unexposed primers were found to contain the inorganic phases SrCrO4, BaSO4 and TiO2 (anatase or rutile). Upon immersion, water uptake by the primers was observed, together with a decrease in the amount of SrCrO4 in the primers. These studies provide insights into the mechanism of chromate leaching from inhibited primers. [Copyright &y& Elsevier]

Birds have become increasingly prominent in studies focusing on natural populations and their coevolved pathogens or examining populations under environmental stress from novel and emerging infectious diseases. For either type of study, new DNA-based diagnostic tests, using the polymerase chain reaction (PCR), present challenges in detecting the DNA of pathogens, which exist in low copy number compared with DNA of the host. One example comes from studies of avian malaria: conflicting claims are made by different laboratories about the accuracy of tests using various sets of primers and reagents, especially in relation to blood smears and immunological methods. There is little standardization of protocol or performance among laboratories conducting tests, in contrast to studies of human malaria. This review compares the problems of detecting avian malaria with those of detecting human malaria, and shows definitively that the buffer used to store blood samples following collection is associated with the accuracy of the test. Lower accuracy is associated with use of a lysis buffer, which apparently degrades the DNA in the blood sample and contributes to inhibition of PCR reactions. DNA extraction and purification techniques, and optimization of the PCR reaction, do not appear to be alternative explanations for the effect of storage buffer. Nevertheless, the purest DNA in standard concentrations for PCR is required so that different primers, DNA polymerases, and diagnostic tests can be objectively compared. [ABSTRACT FROM AUTHOR]

Four microsatellite-enriched DNA libraries yielded 35 microsatellite loci from 100 primer pairs designed for Pacific lion-paw scallop, Nodipecten subnodosus. The number of alleles ranged from four to 28. Three of the 35 loci were not in Hardy–Weinberg equilibrium and linkage disequilibrium was found for one pair of loci. These microsatellites will be used to analyse the population structure of the species in Mexico's Baja Peninsula to propose management strategies for scallop aquaculture development. Twenty-six primer pairs cross-amplified in Nodipecten nodosus, whereas none ( Argopecten ventricosus) or few cross-amplified in the Argopecten species. [ABSTRACT FROM AUTHOR]

Abstract: Comprehensive gene expression analysis of the T cell receptor repertoire of an individual can be very useful in evaluating the immune response in a variety of conditions. Antibody-based analysis methods can detect approximately 60% of the human T cell receptor beta variable (TCRBV) proteins, while gene expression analysis, primarily through employment of the polymerase chain reaction (PCR), has had somewhat greater success in the detection of additional TCRBV families. Many of these previous PCR methods, however, have been unable to detect all 91 alleles of the human TCRBV genes. This is primarily due to either deficiencies in the amplification of all of the variable beta families, subfamilies, and alleles, or the prior lack of a systematic classification of the TCR variable family gene segment sequences. We describe here a real-time reverse transcription polymerase chain reaction-based method, which allows efficient automation and integration of amplification, detection, and analysis with sequence-specific detection of all T cell receptor beta variable gene families, subfamilies, and alleles. This method, which in itself contributes significant improvements over existing technologies through its comprehensiveness and efficiency, also functions independently of variables such as sample source and sample processing and has the ability to run on multiple real-time PCR platforms, affording one the implementation of personal preferences. [Copyright &y& Elsevier]

We developed microsatellite markers for Maculinea nausithous and Maculinea alcon, two of five species of endangered large blue butterflies found in Europe. Two separate microsatellite libraries were constructed. Eleven markers were developed for M. nausithous and one for M. alcon. The primers were tested on both species as well as on the three other European Maculinea species. The number of alleles per locus ranged from two to 14. These markers will be useful tools for population genetic studies of Maculinea species. [ABSTRACT FROM AUTHOR]