Home About us Contact
|
Home About us Contact | ||
|
|
||
Primers Used (primer + used)
Selected AbstractsDirect amplification of nodD from community DNA reveals the genetic diversity of Rhizobium leguminosarum in soilENVIRONMENTAL MICROBIOLOGY, Issue 6 2001Adolphe Zézé Sequences of nodD, a gene found only in rhizobia, were amplified from total community DNA isolated from a pasture soil. The polymerase chain reaction (PCR) primers used, Y5 and Y6, match nodD from Rhizobium leguminosarum biovar trifolii, R. leguminosarum biovar viciae and Sinorhizobium meliloti. The PCR product was cloned and yielded 68 clones that were identified by restriction pattern as derived from biovar trifolii[11 restriction fragment length polymorphism (RFLP) types] and 15 clones identified as viciae (seven RFLP types). These identifications were confirmed by sequencing. There were no clones related to S. meliloti nodD. For comparison, 122 strains were isolated from nodules of white clover (Trifolium repens) growing at the field site, and 134 from nodules on trap plants of T. repens inoculated with the soil. The nodule isolates were of four nodD RFLP types, with 77% being of a single type. All four of these patterns were also found among the clones from soil DNA, and the same type was the most abundant, although it made up only 34% of the trifolii -like clones. We conclude that clover selects specific genotypes from the available soil population, and that R. leguminosarum biovar trifolii was approximately five times more abundant than biovar viciae in this pasture soil, whereas S. meliloti was rare. [source] Evaluating wastewater-induced plant genotoxicity using randomly amplified polymorphic DNAENVIRONMENTAL TOXICOLOGY, Issue 1 2008K. M. Swaileh Abstract Wastewater often contains genotoxic substances that can resist different stages of the treatment process. In the present study, randomly amplified polymorphic DNA technology was applied to evaluate the genotoxic effects of wastewater (treated and raw) irrigation on oat plants (Avena sativa). RAPD profiles obtained showed that both treated and raw wastewater (RWW) were having genotoxic effects on oat plants. This was apparent by the appearance/disappearance of bands in the treatments compared with the control plants. From the 15 primers used, 186 bands were obtained with an average of 12.4 bands per primer. Irrigating plants with RWW caused 51 new bands to appear and 19 to disappear. Treated wastewater (TWW) caused only 16 new bands and the loss of 17 bands. This makes TWW less genotoxic than RWW. The Euclidean distances shown on the dendrogram, revealed the presence of two clusters according to dissimilarity values. One cluster contained the control plants and those irrigated with TWW, whereas the second contained the plants irrigated with RWW. Similarity indices calculated between the treatments and the control plants showed that the control and the plants irrigated with TWW had a similarity index of 0.87, the control and plants irrigated with RWW 0.73 and between the treatments 0.75. © 2008 Wiley Periodicals, Inc. Environ Toxicol, 2008. [source] Direct amplification of rhizobial nodC sequences from soil total DNA and comparison to nodC diversity of root nodule isolatesFEMS MICROBIOLOGY ECOLOGY, Issue 1 2005Sarita Sarita Abstract A group-specific primer set was developed using nodC as a target gene for the amplification of rhizobial sequence diversity from nodule isolates and total soil DNA preparations. The primer set was tested on 209 nodule isolates, recovered from six different trap plant species which were grown in two soil samples collected from a chickpea and a wheat field site in India. We also amplified and cloned PCR products from total DNA isolated from the same soil samples. The total diversity within the resulting clone libraries (, 218 clones) was higher than that recovered from trap plants, but differed depending on the PCR protocols and primers used. However, some plant-selected genotypes could not be obtained using the community approach, probably due to variable detection limits and limited clone library sizes. [source] A combination of baiting and PCR techniques for the detection of Phytophthora quercina and P. citricola in soil samples from oak standsFOREST PATHOLOGY, Issue 2 2001Nechwatal A description is given of the use of a combination of polymerase chain reaction (PCR) and baiting techniques for the specific detection of Phytophthora quercina and Phytophthora citricola from soil around declining oak trees. The soil was flooded with water and subjected to a specific baiting procedure using Quercus robur leaflets as baits. Single round or nested PCR, respectively, with species-specific primers allowed the detection of P. quercina and P. citricola in infected oak leaflets used as baits and in the water from the same bait tests. PCR detection of both fungi was also possible after soil samples had been thoroughly mixed with water and the floating organic debris had been collected. Phytophthora quercina and P. citricola could be readily detected in almost every case in the water from these tests by PCR but less frequently in the organic debris. The identities of P. quercina and P. citricola were confirmed by restriction digests of the corresponding PCR amplicons. The presence of both fungi was also confirmed in parallel in soil samples tested by baiting with oak leaflets. Nested PCR with the primers used allowed the detection of as few as five zoospores of P. citricola and 300 zoospores of P. quercina in a volume of 100 ,l. The methods presented here allow detection and identification of species of Phytophthora in soil without the need for direct extraction of soil samples, and without specific knowledge of the morphological characteristics of the genus. Détection de Phytophthora quercina et de P. citricola dans le sol de chênaies par une méthode combinant le piégeage et la PCR L'article décrit la détection spécifique de Phytophthora quercina et de P. citricola dans le sol prélevé autour de chênes dépérissants, par une méthode combinant les techniques de piégeage et de PCR. Le sol a été immergé dans l'eau et soumis à la procédure du piégeage avec de très jeunes feuilles de Quercus robur. Une amplification par PCR simple ou par PCR gigogne, respectivement, avec des amorces spécifiques des espèces ont permis de détecter P. quercina et P. citricola dans les feuilles-piège infectées, et dans l'eau de piégeage. La détection des deux champignons par PCR a aussi été possible après que les échantillons de sol aient été soigneusement mélangés à de l'eau et que les débris organiques aient été collectés. Phytophthora quercina et P. citricola ont pu être facilement détectés par PCR dans presque tous les cas dans l'eau de piégeage, mais moins fréquemment dans les débris organiques. L'identité de P. quercina et de P. citricola a été confirmée par les profils de restriction des amplifiats obtenus. La présence des deux champignons a aussi été confirmée en parallèle dans des échantillons de sol par piégeage. L'amplification par PCR gigogne avec les amorces utilisées a permis la détection de seulement 5 zoospores de P. citricola et 300 zoospores de P. quercina, dans un volume de 100 ,l. Les méthodes présentées ici permettent la détection et l'identification des espèces de Phytophthora dans le sol en évitant l'extraction directe d'ADN du sol, et sans connaissances spécifiques sur les caractéristiques morphologiques du genre. Eine Kombination von Köder- und PCR-Techniken zum Nachweis von Phytophthora quercina und P. citricola in Bodenproben von Eichenstandorten Es wird der spezifische Nachweis von Phytophthora quercina und P. citricola in Bodenproben von absterbenden Eichen mit Hilfe einer Kombination von PCR- und Baiting-Methoden beschrieben. Die Bodenproben wurden mit Wasser geflutet und Baiting-Tests unterzogen, bei denen junge Blättchen von Quercus robur als Köder zum Einsatz kamen. Einfache oder nested PCR-Reaktionen mit artspezifischen Primern erlaubten den Nachweis von P. quercina und P. citricola in den infizierten Eichenblättchen aus diesen Tests und im jeweiligen ,Baiting-Wasser'. Der PCR-Nachweis beider Erreger war auch möglich, wenn Bodenproben gründlich mit Wasser gemischt wurden, das aufgeschwemmte organische Material abgesammelt und das Wasser abgenommen wurde. P. quercina und P. citricola wurden dabei in nahezu allen Fällen im Wasser, jedoch weniger regelmäßig im organischen Material nachgewiesen. Die Identität der betreffenden Arten wurde zusätzlich durch Restriktions-Analysen der entsprechenden Amplicons bestätigt. Außerdem wurde die Anwesenheit beider Arten in den untersuchten Bodenproben durch klassische Baiting-Methoden nachgewiesen. Nested PCR mit den verwendeten Primerpaaren erlaubte den Nachweis von nur 5 Zoosporen von P. citricola und 300 Zoosporen von P. quercina in einem Gesamt-Volumen von 100 ,l. Die beschriebenen Methoden ermöglichen Nachweis und Identifizierung von Phytophthora-Arten in Bodenproben, ohne die Notwendigkeit einer direkten Extraktion des Bodens und ohne weitreichende Kenntnis der morphologischen Merkmale der Arten dieser Gattung. [source] Automated SEM/EDS Analysis of Airbag Residue.JOURNAL OF FORENSIC SCIENCES, Issue 1 2009II: Airbag Residue as a Source of Percussion Primer Residue Particles Abstract:, Automated scanning electron microscopy with energy dispersive spectroscopy has been used to analyze airbag residue particles. Analysis of airbag residue from some passenger side airbags revealed some residue particles which are consistent with gunshot residue (GSR) samples. The source of these particles was determined to be percussion primers used to initiate the chemical reaction for deployment. This article identifies some vehicles which contain this type of airbag and demonstrates the types of particles which could be misidentified as being GSR. The low numbers of GSR particles in among the large particle populations of zirconium and/or copper,cobalt particles, which are clearly airbag residue, allow the trained analysts to distinguish the correct source of this residue. Particles containing high aluminum levels, elevated levels of allowable elements in GSR particles, or the presence of elements that are rare in GSR particles stand out as indications that the particles are not GSR in origin. This study serves as a guide to analysts who perform particle analysis in forensic investigations. [source] Assessment of Genetic Variation Within Indian Mustard (Brassica juncea) Germplasm Using Random Amplified Polymorphic DNA MarkersJOURNAL OF INTEGRATIVE PLANT BIOLOGY, Issue 4 2008Muhammad Ayub Khan Abstract Genetic diversity among 45 Indian mustard (Brassica juncea L.) genotypes comprising 37 germplasm collections, five advance breeding lines and three improved cultivars was investigated at the DNA level using the random amplified polymorphic DNA (RAPD) technique. Fifteen primers used generated a total of 92 RAPD fragments, of which 81 (88%) were polymorphic. Of these, 13 were unique to accession ,Pak85559'. Each primer produced four to nine amplified products with an average of 6.13 bands per primer. Based on pairwise comparisons of RAPD amplification products, Nei and Li's similarity coefficients were calculated to evaluate the relationships among the accessions. Pairwise similarity indices were higher among the oilseed accessions and cultivars showing narrow ranges of 0.77,0.99. An unweighted pair-group method with arithmetic averages cluster analysis based on these genetic similarities placed most of the collections and oilseed cultivars close to each other, showing a low level of polymorphism between the accessions used. However, the clusters formed by oilseed collections and cultivars were comparatively distinct from that of advanced breeding lines. Genetically, all of the accessions were classified into a few major groups and a number of individual accessions. Advanced breeding lines were relatively divergent from the rest of the accessions and formed independent clusters. Clustering of the accessions did not show any pattern of association between the RAPD markers and the collection sites. A low level of genetic variability of oilseed mustard was attributed to the selection for similar traits and horticultural uses. Perhaps close parentage of these accessions further contributed towards their little diversity. The study demonstrated that RAPD is a simple and fast technique to compare the genetic relationship and pattern of variation among the gene pool of this crop. [source] Screening food products for the presence of CaMV 35S promoter and NOS 3, terminatorJOURNAL OF THE SCIENCE OF FOOD AND AGRICULTURE, Issue 12 2005Hanaa AS Oraby Abstract Biotechnology has enabled the modification of agricultural materials in a very precise way, thereby improving productivity and yields of economically important crops. There are a number of methods available for detecting genetically modified organisms (GMOs). In the present investigation, a qualitative PCR technique has been adopted in order to discriminate between genetically modified and non-modified food products. The qualitative PCR assay employs primers specific for genetic elements that are used to generate genetically engineered agricultural crops. Two of the most common primers used for the detection of GMOs, 35S promoter and NOS 3, terminator, have been tested over a panel of 24 food products purchased from the local market. The results indicated that, out of the 24 food products tested, three products gave positive results with the 35S promoter. The NOS 3, primers gave negative results with all tested samples. Copyright © 2005 Society of Chemical Industry [source] Full genomic amplification and subtyping of influenza A virus using a single set of universal primersMICROBIOLOGY AND IMMUNOLOGY, Issue 3 2010Emi Inoue ABSTRACT Influenza A virus has eight-segmented RNA molecules as a genome and, among all strains of the virus, both ends of each segment have 13 and 12 nucleotide sequences conserved. In the present study, a simple RT-PCR method to amplify all eight segments of the virus and determine the HA and NA subtype using a single primer set based on the conserved terminal sequences has been established. This method is also capable of detecting subgenomic defective interfering RNA of the influenza A virus. Since the primers used here cope with each and every RNA segment of influenza A virus, this simple RT-PCR method is valuable not only for cloning each gene of the virus, but also for identifying subtypes, including subtypes other than 16 HA and 9 NA subtypes. [source] Major histocompatibility complex class II variation in the giant panda (Ailuropoda melanoleuca)MOLECULAR ECOLOGY, Issue 9 2006QIU-HONG WAN Abstract Habitat destruction and human activity have greatly impacted the natural history of the giant panda (Ailuropoda melanoleuca). Although the genetic diversity of neutral markers has been examined in this endangered species, no previous work has examined adaptive molecular polymorphisms in the giant panda. Here, the major histocompatibility complex (MHC) class II DRB locus was investigated in the giant panda, using single-strand conformation polymorphism (SSCP) and sequence analysis. Comparisons of DNA samples extracted from faecal and blood samples from the same individual revealed that the two materials yielded similar quantities and qualities of DNA, as well as identical SSCP patterns and allelic sequences, demonstrating the reliability of DNA isolation from panda faeces. Analysis of faecal samples from 60 giant pandas revealed relatively low number of alleles: seven alleles. However, the alleles were quite divergent, varying from each other by a range of 7,47 nucleotide substitutions (4,25 amino acid substitutions). Construction of a neighbour-joining tree and comparisons among DRB alleles from other species revealed that both similar and highly divergent alleles survived in the bottlenecked panda populations. Despite species-specific primers used and excellent faecal DNA isolated, a lower level of heterozygosity than expected was still observed due to inbreeding. There were three types of evidence supporting the presence of balancing selection in the giant panda: (i) an obvious excess of nonsynonymous substitutions over synonymous at the antigen-binding positions; (ii) trans -species evolution of two alleles between the giant panda and other felids; and (iii) a more even distribution of alleles than expected from neutrality. [source] Distribution of the bacterial symbiont Cardinium in arthropodsMOLECULAR ECOLOGY, Issue 7 2004EINAT ZCHORI-FEIN Abstract ,Candidatus Cardinium', a recently described bacterium from the Bacteroidetes group, is involved in diverse reproduction alterations of its arthropod hosts, including cytoplasmic incompatibility, parthenogenesis and feminization. To estimate the incidence rate of Cardinium and explore the limits of its host range, 99 insect and mite species were screened, using primers designed to amplify a portion of Cardinium 16S ribosomal DNA (rDNA). These arthropods were also screened for the presence of the better-known reproductive manipulator, Wolbachia. Six per cent of the species screened tested positive for Cardinium, compared with 24% positive for Wolbachia. Of the 85 insects screened, Cardinium was found in four parasitic wasp species and one armoured scale insect. Of the 14 mite species examined, one predatory mite was found to carry the symbiont. A phylogenetic analysis of all known Cardinium 16S rDNA sequences shows that distantly related arthropods can harbour closely related symbionts, a pattern typical of horizontal transmission. However, closely related Cardinium were found to cluster among closely related hosts, suggesting host specialization and horizontal transmission among closely related hosts. Finally, the primers used revealed the presence of a second lineage of Bacteroidetes symbionts, not related to Cardinium, in two insect species. This second symbiont lineage is closely allied with other arthropod symbionts, such as Blattabacterium, the primary symbionts of cockroaches, and male-killing symbionts of ladybird beetles. The combined data suggest the presence of a diverse assemblage of arthropod-associated Bacteroidetes bacteria that are likely to strongly influence their hosts' biology. [source] Isolation of Trichosporon in a hematology wardMYCOSES, Issue 1 2005Gabriella Pini Summary During mycologic monitoring of the air in a hematology ward, we found massive air contamination caused by Trichosporon asahii, both in the room where neutropenic patients were staying and the corridor immediately outside the room. This fungal species had never been isolated in previous samplings. The urine culture taken from one of the patients in this room, whose urinary catheter had been removed immediately prior to air sampling, resulted positive for T. asahii. Both macroscopic and microscopic morphologic observation was insufficient for confirming the hypothesis of a close relationship between the strains isolated from the patient, from the air in the room and corridor. Therefore, we used genomic typing with random amplified polymorphic DNA (RAPD). The five primers used, (GTG)5, (GACA)4, M13, OPE01, RC08, produced different patterns of polymerase chain reaction (PCR) products; the genomic profiles obtained with the same primer, however, resulted perfectly superimposable for all the strains. This result led us to conclude that the massive air contamination caused by T. asahii can have effectively been determined by the removal of the urinary catheter from the patient who presented an asymptomatic infection caused by this microorganism. [source] Identification of wheat,Thinopyrum intermedium 2Ai-2 ditelosomic addition and substitution lines with resistance to barley yellow dwarf virusPLANT BREEDING, Issue 2 2006Z. S. Lin Abstract Among the regenerated plants derived from immature hybrid embryos of wheat,Thinopyrum intermedium disomic addition line Z6 × common wheat variety ,Zhong8601', a plant with a telocentric chromosome and barley yellow dwarf virus (BYDV) resistance was obtained. The telocentric chromosome paired with an entire Thinopyrum chromosome to form a heteromorphic bivalent at meiotic metaphase I. Genomic in situ hybridization showed that the telosome originated from Th. intermedium. Two ditelosomic additions and one disomic substitution were identified among the offspring of the plant. Two random amplified polymorphic DNA molecular markers were identified among 150 random primers used to detect the different arms of the alien chromosome. These might be useful for developing translocation lines with BYDV resistance. [source] Multiplex nested-polymerase chain reaction for the simultaneous detection of Aeromonas hydrophila, Edwardsiella tarda, Photobacterium damselae and Streptococcus iniae, four important fish pathogens in subtropical AsiaAQUACULTURE RESEARCH, Issue 10 2009Chin-I Chang Abstract A multiplex nested-polymerase chain reaction (PCR)-based (m-nested PCR) method was developed for simultaneous detection of four important freshwater/marine fish pathogens in subtropical Asia, including Aeromonas hydrophila, Edwardsiella tarda, Photobacterium damselae and Streptococcus iniae. The specificity of the oligonucleotide primers used for PCR detection was confirmed to generate specific amplicons for the corresponding pathogens. Moreover, non-specific amplicons were observed when the primers were tested using pure DNA extracted from 31 related bacterial strains belonging to 23 species or tissue homogenates of infected tilapia. This m-nested PCR approach could detect 19 colony forming unit (CFU) for A. hydrophila, 62 CFU for E. tarda, 280 CFU for P. damselae subsp. piscicida and 179 CFU for S. iniae in infected tilapia kidney homogenates, consistent with the results derived from bacteriological methods. The assay described in this paper is a sensitive and effective method for simultaneous detection of multiple fish pathogens. [source] A new technique of anterior TAP enhances the positivity of CMV by PCR in hypertensives anterior uveitisACTA OPHTHALMOLOGICA, Issue 2009P KOCH Purpose Anterior uveitis can be severely disabling. Especially, hypertensives anterior uveitis can lead to a decrease in visual acuity, posterior synechiaes, cataract, glaucoma, etc. Diagnosis is frequently complex. Two main aetiologies are retained: non infectious (auto-immunes) and infectious forms. Amongst the lasts, various aetiologies are possible. Viral anterior uveitis remained difficult to diagnose for a long time. However, since the emergence of the polymerase chain reaction (PCR), the diagnosis is definitely easier. Nevertheless, anterior TAP result is determined by different limitations including the puncture technique, the PCR primers used, and of course the investigated virus. Methods We hereby propose a new technique of anterior TAP that allowed us to increase our PCR results in CMV anterior uveitis. Two samples were obtained: firstly, a conventional anterior TAP was realised; followed by a rinsing of the anterior chamber with saline solution. A Goldman-Witmer index for rubeola was performed in the first sample. Both samples were examined for viral PCR (HSV1, 2, VZV, CMV, EBV, Rubeola) Results We did not found any side effect of the technique used by comparison with normal anterior TAP. Diagnosis was obtained in 20 of the 35 eyes tested. Rubeola diagnosis was obtained in 11/20 eyes, VZV in 1/20, HSV1 in 4/20, and CMV in 4/20. Intriguingly, CMV diagnosis was obtained in three cases only in the second syringe and not in the first Conclusion We have, to date, detected 4 cases of CMV anterior uveitis in a cohort of 35 patients with anterior uveitis. We did not meet any complication but obtained interesting results concerning CMV diagnosis, using a rinsing of the anterior chamber (second syringe). [source] | |||||||||||||||||||