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Nested Polymerase Chain Reaction (nested + polymerase_chain_reaction)
Selected AbstractsDetection of Puccinia striiformis in Latently Infected Wheat Leaves by Nested Polymerase Chain ReactionJOURNAL OF PHYTOPATHOLOGY, Issue 7-8 2009Xiaojie Wang Abstract Stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is one of the most devastating wheat diseases worldwide, especially in temperate regions with cool moist weather conditions. The identification of the pathogen in infected plants based on morphological or physiological criteria before sporulation is labour-intensive and time-consuming. To accelerate and simplify the process of detection, a nested Polymerase Chain Reaction (PCR) assay was developed for specific and sensitive detection of Pst. Specific primers Psta-Psts were designed according to a genome-specific sequence of Pst. In nested PCR, with a 10-fold dilution series of template DNA, the detection limit was 2 pg DNA in the first PCR with the primers Psta-Psts. The second round PCR was then performed using amplified products from the first PCR as the template and Nesta-Nests as the primers. An amplification signal was detectable even when only 2 fg of P. striiformis f. sp. tritici DNA was used as the template in nested PCR. With nested PCR, the sensitivity of detection was enhanced 1000 fold. Using extracts from stripe rust-infected wheat leaves, the fungus could be determined in the leaves before symptom appearance. The assay provides a rapid and sensitive method for detection of P. striiformis f. sp. tritici in latently infected leaves of overwintering wheat plants. [source] Molecular Characterization and Potential Insect Vector of a Phytoplasma Associated with Garden Beet Witches' Broom in Yazd, IranJOURNAL OF PHYTOPATHOLOGY, Issue 4 2007A. Mirzaie Abstract In 2002, garden beet witches' broom (GBWB) phytoplasma was detected for the first time in garden beet plants (Beta vulgaris L. ssp. esculenta) in Yazd, Iran. Nested polymerase chain reaction (PCR) and restriction fragment length polymorphic (RFLP) analysis of PCR-amplified phytoplasma 16S rDNA were employed for the detection and identification of the phytoplasma associated with garden beet. A phytoplasma belonging to subgroup 16SrII-E, in the peanut witches' broom group (16SrII), was detected in infected plants. Asymptomatic plant samples and the negative control yielded no amplification. The result of analysis of the nucleotide sequence of a 1428 bp fragment of 16S rDNA gene from GBWB phytoplasma (GenBank accession number DQ302722) was basically consistent with the classification based on RFLP analysis, in which GBWB phytoplasma clustered with phytoplasmas of the 16SrII-E subgroup. A search for a natural phytoplasma vector was conducted in Yazd in 2004, in an area where garden beet crops had been affected since 2002. The associated phytoplasma was detected in one leafhopper species, Orosius albicinctus, commonly present in this region. The leafhopper O. albicinctus was used in transmission tests to determine its vector status for the phytoplasma associated with GBWB. Two of eight plants that had been fed on by O. albicinctus, showed mild symptoms of GBWB including stunting and reddening of midveins. A phytoplasma was detected in the two symptomatic test plants by PCR using universal primers and it was identified by RFLP as the GBWB phytoplasma. This finding suggests O. albicinctus is a vector of the GBWB phytoplasma. [source] Detection of Puccinia striiformis in Latently Infected Wheat Leaves by Nested Polymerase Chain ReactionJOURNAL OF PHYTOPATHOLOGY, Issue 7-8 2009Xiaojie Wang Abstract Stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is one of the most devastating wheat diseases worldwide, especially in temperate regions with cool moist weather conditions. The identification of the pathogen in infected plants based on morphological or physiological criteria before sporulation is labour-intensive and time-consuming. To accelerate and simplify the process of detection, a nested Polymerase Chain Reaction (PCR) assay was developed for specific and sensitive detection of Pst. Specific primers Psta-Psts were designed according to a genome-specific sequence of Pst. In nested PCR, with a 10-fold dilution series of template DNA, the detection limit was 2 pg DNA in the first PCR with the primers Psta-Psts. The second round PCR was then performed using amplified products from the first PCR as the template and Nesta-Nests as the primers. An amplification signal was detectable even when only 2 fg of P. striiformis f. sp. tritici DNA was used as the template in nested PCR. With nested PCR, the sensitivity of detection was enhanced 1000 fold. Using extracts from stripe rust-infected wheat leaves, the fungus could be determined in the leaves before symptom appearance. The assay provides a rapid and sensitive method for detection of P. striiformis f. sp. tritici in latently infected leaves of overwintering wheat plants. [source] Angiotensin-I-converting enzyme insertion/deletion polymorphism and high urinary albumin concentration in French Type 2 diabetes patientsDIABETIC MEDICINE, Issue 8 2003S. Hadjadj Abstract Aims Family-based studies suggest a genetic basis for nephropathy in Type 2 diabetes. The angiotensin-I-converting enzyme (ACE) gene is a candidate gene for Type 1 diabetes nephropathy. We assessed the association between high urinary albumin concentration and ACE insertion/deletion (I/D) polymorphism, in French Type 2 diabetes patients. Methods We studied 3139 micro/macroalbuminuric French patients recruited in the DIABHYCAR Study, an ACE inhibition trial in Type 2 diabetes patients with renal and cardiovascular outcomes. The main inclusion criteria were age , 50 years, urinary albumin concentration , 20 mg/l assessed centrally during two consecutive screening visits, and plasma creatinine concentration , 150 µmol/l. These patients were compared with 605 normoalbuminuric (NA; urinary albumin concentration < 10 mg/l at first screening for the DIABHYCAR Study) French patients. ACE I/D genotype was determined by nested polymerase chain reaction. Results The ACE I/D polymorphism was in Hardy,Weinberg equilibrium. The distribution of genotypes did not differ significantly between micro/macroalbuminuric and NA patients: 552 and 115 II, 1468 and 282 ID, 1119 and 208 DD (P = 0.67). However, the ACE D allele was more frequent among normotensive micro/macroalbuminuric patients than among NA patients (P = 0.039). Conclusions The ACE I/D polymorphism was not associated with high urinary albumin concentration in French Type 2 diabetes patients. [source] Human enteric viruses in groundwater indicate offshore transport of human sewage to coral reefs of the Upper Florida KeysENVIRONMENTAL MICROBIOLOGY, Issue 4 2010J. Carrie Futch Summary To address the issue of human sewage reaching corals along the main reef of the Florida Keys, samples were collected from surface water, groundwater and coral [surface mucopolysaccharide layers (SML)] along a 10 km transect near Key Largo, FL. Samples were collected semi-annually between July 2003 and September 2005 and processed for faecal indicator bacteria (faecal coliform bacteria, enterococci and Clostridium perfringens) and human-specific enteric viruses (enterovirus RNA and adenovirus DNA) by (RT)-nested polymerase chain reaction. Faecal indicator bacteria concentrations were generally higher nearshore and in the coral SML. Enteric viruses were evenly distributed across the transect stations. Adenoviruses were detected in 37 of 75 samples collected (49.3%) whereas enteroviruses were only found in 8 of 75 samples (10.7%). Both viruses were detected twice as frequently in coral compared with surface water or groundwater. Offshore, viruses were most likely to be found in groundwater, especially during the wet summer season. These data suggest that polluted groundwater may be moving to the outer reef environment in the Florida Keys. [source] Isolation and gene quantification of heterotrophic N2 -fixing bacterioplankton in the Baltic SeaENVIRONMENTAL MICROBIOLOGY, Issue 1 2007Kjärstin H. Boström Summary Cyanobacteria are regarded as the main N2 -fixing organisms in marine waters. However, recent clone libraries from various oceans show a wide distribution of the dinitrogenase reductase gene (nifH) originating from heterotrophic bacterioplankton. We isolated heterotrophic N2 -fixing bacteria from Baltic Sea bacterioplankton using low-nitrogen plates and semi-solid diazotroph medium (SSDM) tubes. Isolates were analysed for the nitrogenase (nifH) gene and active N2 fixation by nested polymerase chain reaction (PCR) and acetylene reduction respectively. A primer-probe set targeting the nifH gene from a , - proteobacterial isolate, 97% 16S rDNA similarity to Pseudomonas stutzeri, was designed for measuring in situ dynamics using quantitative real-time PCR. This nifH gene sequence was detected at two of 11 stations in a Baltic Proper transect at abundances of 3 × 104 and 0.8 × 103 copies per litre seawater respectively. Oxygen requirements of isolates were examined by cultivation in SSDM tubes where oxygen gradients were determined with microelectrodes. Growth, and thereby N2 fixation, was observed as horizontal bands formed at oxygen levels of 0,6% air saturation. The apparent microaerophilic or facultative anaerobic nature of the isolates explains why the SSDM approach is the most appropriate isolation method. Our study illustrates how combined isolation, functional analyses and in situ quantification yielded insights into the oxygen requirements of heterotrophic N2 -fixing bacterioplankton isolates, which were confirmed to be present in situ. [source] Identification of fruit tree phytoplasmas and their vectors in Bosnia and HerzegovinaEPPO BULLETIN, Issue 2 2007D. Delic Surveys were carried out in autumn 2004 and spring 2005 in the traditional areas dedicated to pome and stone fruit cultivation in Bosnia and Herzegovina to assess the presence, distribution and incidence of phytoplasma diseases in fruit trees. The occurrence of psyllid vectors was also considered. The detection of phytoplasmas in plant and insect samples and their identification were carried out by symptom observations in the field, double antibody sandwich-enzyme linked immunosorbent assay (DAS-ELISA), nested polymerase chain reaction (nested-PCR) and restriction fragment length polymorphism (RFLP) analyses. Laboratory analyses showed the presence of phytoplasmas belonging to: (i) 16SrX group, subgroup A (,Candidatus Phytoplasma mali') in 23 out of 25 apple samples, in 4 groups out of 18 of Cacopsylla picta (synonym Cacopsylla costalis) and in 2 groups out of 9 of Cacopsylla melanoneura; (ii) 16SrX group, subgroup C (,Candidatus Phytoplasma pyri') in 11 out of 30 pears samples and in 2 groups out of 9 of Cacopsylla pyri; (iii) 16SrX group, subgroup B (,Candidatus Phytoplasma prunorum') in 4 apricots, 2 peaches out of 42 stone fruit samples and in 1 group out of 14 of Cacopsylla pruni. The presence of different subtypes of Candidatus Phytoplasma mali, both in apple trees and in insects, was proven. [source] Analysis of the distribution of Phytophthora cinnamomi in soil at a disease site in Western Australia using nested PCRFOREST PATHOLOGY, Issue 2 2009N. Williams Summary The oomycete plant pathogen Phytophthora cinnamomi has infected a very large area of native vegetation in the south western corner of Australia. An important aspect of effective disease management depends on being able to accurately map areas of infestation. For this purpose, we have developed a nested polymerase chain reaction (PCR) protocol for the detection of P. cinnamomi in soil. The test uses two sets of primers developed from the rRNA ITS sequences of P. cinnamomi and can detect as little as 1 pg DNA. The degree of sensitivity was reduced with DNA extracted from soil although this depended on the type of soil. Soils with a high organic content, such as eucalypt forest soil and potting mix were more inhibitory than sandy soils. Inhibition by soil DNA could be reduced by the addition of bovine serum albumin and formamide to the reaction. Taq DNA polymerase was very sensitive to inhibitors compared with Tth+ or TaqF1*. In comparison with baiting (0,10% positive samples), nested PCR proved to be a very much more efficient (90,100% positive samples) method for the detection of P. cinnamomi in soil. [source] Multiple Displacement Amplification of Isolated DNA from Human Gallstones: Molecular Identification of Helicobacter DNA by Means of 16S rDNA-Based Pyrosequencing AnalysisHELICOBACTER, Issue 6 2005Isabelle Nilsson ABSTRACT Background., Molecular typing of Helicobacter spp. in clinical biopsy specimens has become increasingly important. By means of nested polymerase chain reaction (PCR) amplification and Southern blot analysis of the PCR amplicons, we have shown that Helicobacter spp. DNA is present in human gallstones. In this study we have investigated the possibility of using multiple displacement amplification (MDA) of isolated gallstone DNA and pyrosequencing analysis for the molecular identification of Helicobacter spp. Materials and Methods., DNA isolated from the nucleus of 33 human gallstones and one control strain were used in a MDA assay. Subsequently, pyrosequencing analysis was performed either directly on MDA-DNA using primers flanking the Helicobacter spp. 16S rDNA variable V3 region or on PCR amplicons derived from broad-range primers flanking the 16S rDNA variable V3, V4, and V9 regions. Results., Pyrosequencing analysis of 16S rDNA derived from MDA-DNA revealed that Helicobacter spp.-like DNA was present in 25 of 33 (approximately 76%) gallstones. Using an H. pylori- specific Southern blot analysis, Helicobacter spp.-like DNA was present in 20 of 33 [approximately 61%] of the gallstones. Using MDA-DNA directly in pyrosequencing analysis, Helicobacter spp.-like DNA was present in 13 of 33 [approximately 39%] gallstones. Conclusions., We conclude that multiple displacement amplification combined with pyrosequencing enables a rapid and accurate molecular typing of Helicobacter spp. from small and precious biopsy specimens. [source] Evolution of multi-drug resistant hepatitis B virus during sequential therapy,HEPATOLOGY, Issue 3 2006Hyung Joon Yim Multi-drug resistant hepatitis B virus (HBV) has been reported in hepatitis B patients who received sequential antiviral therapy. In vitro studies showed that HBV constructs with mutations resistant to lamivudine and adefovir have marked reduction in sensitivity to combination of lamivudine and adefovir, whereas constructs with mutations resistant to either drug remain sensitive to the other drug. We conducted this study to determine whether mutations conferring resistance to multiple antiviral agents co-locate on the same HBV genome in vivo and to describe the evolution of these mutations. Sera from six patients who had been found to have multi-drug resistant HBV mutations to lamivudine + adefovir, lamivudine + hepatitis B immunoglobulin (HBIG), or lamivudine + entecavir on direct sequencing were cloned after nested polymerase chain reaction (PCR). Analysis of 215 clones from 11 samples with multi-drug resistant mutations on direct sequencing showed that 183 (85%) clones had mutations to both therapies on the same genome; 31 clones had lamivudine-resistant mutants only. Clonal analysis of serial samples from three patients showed progressive evolution from all clones with lamivudine-resistant HBV mutations only to mixtures of clones that have multi-drug resistant mutations and clones that have lamivudine-resistant HBV mutations only, and ultimately all clones having multi-drug resistant HBV mutations. In conclusion, mutations conferring resistance to multiple antiviral agents co-locate on the same viral genome, suggesting that combination therapy directed against mutants resistant to each treatment may not be adequate in suppressing multi-drug resistant HBV. De novo combination therapy may prevent the emergence of multi-drug resistant mutants. (HEPATOLOGY 2006;44:703,712.) [source] An optimized nested polymerase chain reaction (PCR) approach allows detection and characterization of human immunodeficiency virus type 1 (HIV-1) env and gag genes from clinical samplesJOURNAL OF CLINICAL LABORATORY ANALYSIS, Issue 2 2008Dayse Locateli Abstract The needs for development and/or improvement of molecular approaches for microorganism detection and characterization such as polymerase chain reaction (PCR) are of high interest due their sensitivity and specificity when compared to traditional microbiological techniques. Considering the worldwide importance of human immunodeficiency virus type 1 (HIV-1) infection, it is essential that such approaches consider the genetic variability of the virus, the heterogeneous nature of the clinical samples, the existence of contaminants and inhibitors, and the consequent needs for standardization in order to guarantee the reproducibility of the methods. In this work we describe a nested PCR assay targeting HIV-1 virus gag and env genes, allowing specific and sensitive diagnosis and further direct characterization of clinical samples. The method described herein was tested on clinical samples and allowed the detection of HIV-1 presence in all samples tested for the gag gene and 90.9% for the env gene, revealing sensitivities of 1,fg and 100,fg, respectively. Also, no cross-reactions were observed with DNA from infected and noninfected patients and the method allowed detection of the env and gag genes on an excess of 108 and 104 of human deoxyribonucleic acid (DNA), respectively. Furthermore, it was possible to direct sequence all amplified products, which allowed the sub typing of the virus in clinical samples. J. Clin. Lab. Anal. 22:106,113, 2008. © 2008 Wiley-Liss, Inc. [source] Multiple human papillomavirus DNA identified in verruciform xanthoma by nested polymerase chain reaction with degenerate consensus primersJOURNAL OF CUTANEOUS PATHOLOGY, Issue 5 2003Angela Rohwedder First page of article [source] Larvae and early post-larvae of Penaeus monodon (Fabricius) experimentally infected with white spot syndrome virus (WSSV) show no significant mortalityJOURNAL OF FISH DISEASES, Issue 7 2003K Yoganandhan Abstract The pathogenicity of white spot syndrome virus (WSSV) was tested with different developmental stages of Penaeus monodon, i.e. nauplius, protozoeae, mysis, early post-larvae (PL1-10), late post-larvae (PL11-20) and juveniles. WSSV challenge was done by immersion and oral routes. No disease occurred in the larvae and early post-larvae but they were positive for WSSV by nested polymerase chain reaction (PCR) assay. Significant mortality was observed in late post-larvae and juveniles and both single and nested PCR assays gave positive results with these samples. The results demonstrated that WSSV virulence in P. monodon increases with advancing stages of development and that WSSV infection does not result in disease for larvae and post-larvae younger than PL10. [source] Occult hepatitis B virus infection in Southern African blacks with hepatocellular carcinomaJOURNAL OF GASTROENTEROLOGY AND HEPATOLOGY, Issue 9 2008Michael C Kew Abstract Background and Aim:, To ascertain the prevalence of occult hepatitis B virus (HBV) infection in southern African blacks with hepatocellular carcinoma. Methods:, Sera from 118 patients negative for HBV surface antigen but positive for HBV antibodies were studied. HBV-DNA was detected using a nested polymerase chain reaction (PCR) assay and confirmed by nucleotide sequencing of the surface and precore/core genes. Results:, Surface gene HBV-DNA was detected in a single PCR assay in 48.4% of the patients. Positive results increased to 57.7% after two PCR assays (not significant) and 75.7% after four assays (P < 0.001). No false positive results were obtained in these assays or in the 15 control samples for which PCR assays were performed four times. Significant differences in positivity rates were not observed between patients positive for HBV core antibody alone and those positive for core and surface antibodies. The sensitivity of the PCR amplification of the precore/core gene was significantly less than that of the surface open reading frame: the yield of positive results was 23.7% after one assay, 32.2% after two assays (not significant), and 52% after four assays (P < 0.001). Combining the results of the assays of the two genes increased the yield of positive results for the first assay (by 11.9%, P = 0.015), but not the second (6.1%) or fourth assays (4.6%). Conclusion:, Occult HBV infection is present in the serum of the majority of hepatocellular carcinomas in southern African blacks whose serum is negative for hepatitis B surface antigen but positive for anti-HBV core antigen. The yield of positive results increases if more than one PCR assay is performed. [source] Yersinia pseudotuberculosis infection in breeding monkeys: detection and analysis of strain diversity by PCRJOURNAL OF MEDICAL PRIMATOLOGY, Issue 3 2002T. Kageyama In the last three decades, several monkeys reared in outdoor/indoor,outdoor breeding colonies and cages of the Primate Research Institute, Kyoto University, died of yersiniosis caused by Yersinia pseudotuberculosis, necessitating introduction of a method to detect the bacteria rapidly and thus allow preventive measures to be undertaken. A rapid nested polymerase chain reaction (PCR) method for identification of Y. pseudotuberculosis in fecal samples and a random amplified polymorphic DNA (RAPD)-PCR approach for distinguishing between bacterial strains were therefore developed. Yersinia pseudotuberculosis isolates from monkey specimens were found to be classifiable into several types. To determine the source of infection, hundreds of fecal samples of wild rats, pigeons, and sparrows were collected from around the breeding colonies and cages, and subjected to PCR analyses. Yersinia pseudotuberculosis was detected in 1.7% of the fecal samples of wild rats. The DNA fingerprints of the bacteria revealed by RAPD-PCR were the same as that of one strain isolated from macaques, suggesting the wild rat to be a possible source of infection. [source] Seroprevalence of human T-lymphotropic virus type 1 and 2 in Korean blood donorsJOURNAL OF MEDICAL VIROLOGY, Issue 10 2008So-Yong Kwon Abstract The seroprevalence rate of human T-lymphotropic virus (HTLV) among the Korean blood donor population was studied to determine whether screening for HTLV should be implemented. A total of 15,173 serum samples collected from June to July 2006 at five Blood Centers which are located closely to Japan geographically, where the prevalence of HTLV is known to be high, were tested. Serological screening was done by a chemiluminescence method. Samples reactive repeatedly on serological screening were confirmed further by Western blot, line immunoassay, nested polymerase chain reaction and sequencing of proviral DNA. Six samples tested reactive with the serological assay showing a reactive rate of 0.004%. Among the six samples, one sample was confirmed as HTLV-1 positive, giving a confirmed reactive rate of 0.007%. Based on the results of this study, an extended study will be conducted to evaluate whether introduction of HTLV screening is necessary in Korea. J. Med. Virol. 80:1864,1867, 2008. © 2008 Wiley-Liss, Inc. [source] Characterization of Phytoplasmas Associated With Almond Diseases in IranJOURNAL OF PHYTOPATHOLOGY, Issue 11-12 2009L. Zirak Abstract In recent years, almond witches'-broom disease has been prevalent in almond growing areas in the centre and south of Iran. Furthermore, almond trees showing different symptoms of phytoplasma diseases such as little leaf, leaf rolling, dieback of branches, rosette and yellowing were observed in the central regions of Iran. DNA isolated from symptomatic almond trees was used to amplify 16S rDNA and 16S-23S rDNA intergenic spacer (IS) fragments by nested polymerase chain reaction (PCR) using phytoplasma universal primer pairs (P1/P7, R16F2/R2, PA2F/R and NPA2F/R). Phytoplasmas were detected in symptomatic almonds in two major almond-growing regions, Isfahan and ChaharMahal-O-Bakhtiari. Restriction fragment length polymorphism analyses of nested PCR products using endonuclease enzymes HpaII and TaqI revealed that phytoplasmas associated with infected almonds are genetically different. Sequence analyses of amplified fragments of 16S rDNA and IS region indicated that the almond phytoplasmas in Iran are closely related to ,Candidatus (Ca.) Phytoplasma aurantifolia', ,Ca. Phytoplasma phoenicium', ,Ca. Phytoplasma solani' and ,Ca. Phytoplasma trifolii'. The phytoplasmas related to ,Ca. Phytoplasma aurantifolia' were more prevalent than other phytoplasmas in the central regions of Iran. [source] Detection of Phytoplasma Infection in Rose, with Degeneration SymptomsJOURNAL OF PHYTOPATHOLOGY, Issue 1 2001M. Kami In 1998 a severe disease was observed on rose cvs. ,Patina', ,Papillon' and ,Mercedes' cultivated in a commercial greenhouse in Poland. The symptoms included stunted growth, bud proliferation, leaf malformation and deficiency of flower buds. Sporadically some plants yielded flower buds transformed into big-bud structures and degenerated flowers. The presence of phytoplasma in roses with severe symptoms as well as in recovered plants and Catharanthus roseus experimentally infected by grafting and via dodder was demonstrated by nested polymerase chain reaction assay with primers pair R16F2/R2 or R16F1/R0 and R16(I)F1/R1 amplifying phytoplasma 16S rDNA fragment. The polymerase chain reaction products (1.1 kb) used for restriction fragment length polymorphism analysis after digestion with endonuclease enzymes AluI and MseI produced the same restriction profiles for all samples. The restriction profiles of phytoplasma DNA from these plants corresponded to those of an aster yellows phytoplasma reference strain. Electron microscope examination of the ultra-thin sections of the stem showed wall thickenings of many sieve tubes of the diseased roses and single phytoplasma cells within a sieve element of the phloem of experimentally infected periwinkles. This paper is the first report on aster yellows phytoplasma in rose identified at a molecular level. Detektion einer Phytoplasma-Infektion bei Rosen mit Degenerationserscheinungen Im Jahr 1998 wurde eine schwere Krankheit bei Rosen der Sorten ,Patina', ,Papillon' und ,Mercedes' festgestellt, die in einem polnischen Gewächshaus für kommerzielle Zwecke kultiviert wurden. Zu den Symptomen gehörten Kümmerwuchs, durchwachsene Knospen, Blattmißbildungen und ein Mangel an Blütenknospen. Einige wenige Pflanzen trugen übergroße Blütenknospen, die degenerierte Blüten hervorbrachten. Die Anwesenheit von Phytoplasmen in Rosen mit starken Symptomen, in erholten Pflanzen und in Catharanthus roseus, der durch Pfropfen und durch Teufelszwirn (Cuscuta) experimentell infiziert worden war, wurde mittels einer genesteten Polymerase-Kettenreaktion mit den Primerpaaren R16F2/R2 oder R16F1/R0 und R16(1)F1/R1 zur Amplifikation des Phytoplasma-16S rDNA-Fragments demonstriert. Die für die Analyse der Restriktionsfragmentlängenpolymorphismen nach Verdau mit den Endonucleasen AluI und MseI verwendeten PCR-Produkte (1,1 kb) produzierten bei allen Proben die gleichen Restriktionsprofile. Die Restriktionsprofile der Phytoplasma-DNA aus diesen Pflanzen entsprachen denjenigen eines Typenstamms eines Asternvergilbung auslösenden Phytoplasmas. Elektronenmikroskopische Untersuchungen ultradünner Schnitte des Stamms zeigten Wandverdickungen bei zahlreichen Siebröhren der erkrankten Rosen und einzelne Phytoplasmazellen innerhalb eines Siebelements des Phloems experimentell infizierter Immergrün-Pflanzen. Dies ist der erste Bericht über ein auf molekularer Ebene identifiziertes Asternvergilbungs-Phytoplasma bei Rosen. [source] Subclinical reactivation of herpes simplex virus type 1 in the oral cavityMOLECULAR ORAL MICROBIOLOGY, Issue 5 2000B. Knaup Reactivation in the oral cavity either symptomatically (recrudescence) or without symptoms (recurrence) may contribute to the transmission of herpes simplex virus type 1 (HSV-1), especially in critical areas of exposure such as dentistry. In order to measure the frequency of HSV-1 reactivation, nested polymerase chain reaction (PCR) was performed on oral swabs collected from 30 healthy people over a period of 58,161 days. In total 19 of 25 (76%) seropositive people were PCR-positive at least once, 6 of these 19 (32%) had recrudescence and 13 (68%) had only asymptomatic reactivation. Frequencies of additional recurrences were higher in people showing symptomatic reactivation than in those who had only recurrences. Recrudescence is a risk factor for elevated levels of asymptomatic HSV-shedding. In most cases HSV-1 was detected only by nested PCR investigated by early onset of therapy or time span before sampling. [source] Genetic diversity of the arbuscular mycorrhizal fungus Glomus intraradices as determined by mitochondrial large subunit rRNA gene sequences is considerably higher than previously expectedNEW PHYTOLOGIST, Issue 2 2008Boris Börstler Summary ,,Glomus intraradices is a widespread arbuscular mycorrhizal fungus (AMF), which has been found in an extremely broad range of habitats, indicating a high tolerance for environmental factors and a generalist life history strategy. Despite this ecological versatility, not much is known about the genetic diversity of this fungal species across different habitats or over large geographic scales. ,,A nested polymerase chain reaction (PCR) approach for the mitochondrial rRNA large subunit gene (mtLSU), distinguished different haplotypes among cultivated isolates of G. intraradices and within mycorrhizal root samples from the field. ,,From analysis of 16 isolates of this species originating from five continents, 12 mitochondrial haplotypes were distinguished. Five additional mtLSU haplotypes were detected in field-collected mycorrhizal roots. Some introns in the mtLSU region appear to be stable over years of cultivation and are ancestral to the G. intraradices clade. ,,Genetic diversity within G. intraradices is substantially higher than previously thought, although some mtLSU haplotypes are widespread. A restriction fragment length polymorphism approach also was developed to distinguish mtLSU haplotypes without sequencing. Using this molecular tool, intraspecific genetic variation of an AMF species can be studied directly in field plants. [source] Genital Carriage of Human Papilloma Virus (HPV) DNA in Prepubertal Girls with and without Vulval DiseasePEDIATRIC DERMATOLOGY, Issue 3 2003Jennifer Powell, M.R.C.P. Our objective was to compare HPV in prepubertal girls with and without lichen sclerosus (LS). We compared the frequencies and types of HPV in girls with LS with those in children with non-LS vulval disease (vulval swab and urine) and in children with no known vulval disease (urine only). HPV DNA was detected using a nested polymerase chain reaction (PCR) with general and consensus primers amplifying a region of the L1 gene, and PCR amplicons were typed using reverse hybridization with labeled HPV type-specific probes. Specimens untypeable by this method were typed by DNA sequencing. In the cohort of children with LS, we recorded the presence of maternal anogenital warts or a dysplastic cervical smear within 3 years of the affected child's birth. We found that HPV was present in the urine and vulval swabs of 8 of 32 children with LS and in 2 of 31 children with non-LS vulval disease, but also in the urine of 7 of 29 controls. In those with LS, the frequency was not increased significantly, but the types were predominantly those commonly associated with dysplasia of the cervix, penis, vulva, and anus, as opposed to the broader spectrum of types found in the control group, not all dysplasia associated. Two of the 32 mothers reported warts, and 15 of 32 (46.9%) had an abnormal smear. (The national average of abnormal cervical smears is less than 10%.) We concluded that HPV appears to be common in all prepubertal girls, but children with LS carried types associated with dysplasia and their mothers had had a high incidence of dyskaryotic smears. [source] Nested PCR-RFLP is a high-speed method to detect fungicide-resistant Botrytis cinerea at an early growth stage of grapesPEST MANAGEMENT SCIENCE (FORMERLY: PESTICIDE SCIENCE), Issue 2 2009Seiya Saito Abstract BACKGROUND: Grey mould caused by the fungus Botrytis cinerea Pers. ex Fr. is one of the major diseases in grapes. The use of fungicides is a simple strategy to protect grapes against B. cinerea disease. However, phenotypes exhibiting resistance to fungicides have been detected in B. cinerea populations. The variation of fungicide-resistant B. cinerea isolates renders B. cinerea disease control difficult in grapevine fields. RESULTS: The authors have developed a nested polymerase chain reaction,restriction fragment length polymorphism (PCR-RFLP) method to detect fungicide-resistant B. cinerea isolates at an early growth stage of grapes in grapevine fields. The nested PCR-RFLP method was carried out to detect benzimidazole-, phenylcarbamate- and/or dicarboximide-resistant B. cinerea isolates from grape berries and leaves at Eichorn,Lorenz growth stage 25 to 29. This method successfully detected fungicide-resistant B. cinerea isolates at an early growth stage of grapes. In addition, only 8 h was required from tissue sampling to phenotyping of fungicide resistance of the isolates. CONCLUSION: It is proposed that the early diagnosis of fungicide-resistant B. cinerea isolates would contribute to further improvement of integrated pest management against B. cinerea in grapevine fields, and that the nested PCR-RFLP method is a high-speed, sensitive and reliable tool for this purpose. Copyright © 2008 Society of Chemical Industry [source] Sensitive detection of Ralstonia solanacearum in soil: a comparison of different detection techniquesPLANT PATHOLOGY, Issue 4 2000P. M. Pradhanang The sensitivity and specificity of various methods were compared for routine detection of Ralstonia solanacearum in a sandy loam soil. Populations fewer than 102 CFU per g soil were detected by dilution plating on a modified semiselective medium (SMSA). In comparison, a tomato bioassay was shown consistently to detect populations at or greater than 7·5 × 105 CFU per g soil. An indirect enzyme-linked immunosorbent assay (ELISA) was as sensitive as the tomato bioassay, but detected as few as 104 CFU per g soil when the suspension was first incubated in SMSA broth prior to testing. Detection using a nested polymerase chain reaction (PCR) was equally as sensitive as that using culture on SMSA agar, but only when the infested soil sample was first enriched overnight in SMSA broth prior to the nested PCR. Longer incubation periods in SMSA broth also increased the sensitivity of pathogen detection using a conventional PCR method, permitting detection of as few as 102 CFU per g soil after 60 h enrichment in SMSA broth. When evaluated using naturally infected field soils in Nepal, isolation of R. solanacearum on SMSA was reliable only when pathogen populations were higher than those of saprophytic soilborne bacteria. As few as 5 × 102 CFU of R. solanacearum per g were recovered from naturally infested soil, whereas the sensitivity of indirect ELISA was 106 CFU g,1. [source] Delayed acquisition of somatic hypermutations in repopulated IGD+CD27+ memory B cell receptors after rituximab treatmentARTHRITIS & RHEUMATISM, Issue 8 2009Khalid Muhammad Objective Transient B cell depletion by rituximab has been used with clinical efficacy in the treatment of patients with rheumatoid arthritis (RA). Previous studies of B cell repopulation have shown long-term numerical reduction in memory B cells. Non,class-switched IgD+CD27+ memory B cells, in particular, repopulate slowly. This study was undertaken to determine whether mutational acquisition in individual B cell receptors in repopulating class-switched and non,class-switched memory B cells is affected by rituximab. Methods Cells obtained from 16 RA patients, 4 healthy donors, and 3 patients who underwent allogeneic stem cell transplantation (ASCT) were analyzed using single B cell sorting followed by nested polymerase chain reaction and Ig VH3 sequencing. Results There was a delayed acquisition of mutations in Ig receptors of IgD+ memory B cells over a period of 6 years after a single course of rituximab. One year after rituximab treatment, 84% of single repopulating IgD+CD27+ B cells were unmutated, and no highly mutated Ig receptors were found (compared with 52% before therapy). Over time, increasing numbers of mutations were detected. Even 6 years after rituximab treatment, however, mutations in IgD+ memory B cells were still significantly reduced. In contrast, class-switched memory B cells repopulated with quantitatively normal mutations. In comparison, in patients undergoing ASCT, IgD+ memory cells repopulated earlier with higher mutations in Ig receptors. Conclusion Our data suggest that IgD+ memory B cells are particularly susceptible to the effects of rituximab, with delayed acquisition of mutations in their Ig receptors still evident 6 years after a single course of rituximab. Our findings indicate that these cells have different requirements for mutational acquisition compared with class-switched memory B cells. [source] Molecular detection of dermatophytes and nondermatophytes in onychomycosis by nested polymerase chain reaction based on 28S ribosomal RNA gene sequencesBRITISH JOURNAL OF DERMATOLOGY, Issue 5 2009M. Ebihara Summary Background, Onychomycosis is often caused by dermatophytes, but the role of nondermatophytes is underestimated due to the difficulty of identifying them by conventional direct microscopy and culture. Objectives, This study aims to detect nondermatophytes, as well as dermatophytes, in the nail samples of patients with onychomycosis using a polymerase chain reaction (PCR)-based culture-independent method. Materials and methods, The nested PCR assay targeting the sequence of the 28S ribosomal RNA gene was used to amplify fungal DNAs from 50 microscopy-positive nail specimens. Newly designed primer sets for dermatophyte universal, Trichophyton rubrum, T. mentagrophytes, Aspergillus spp., Scopulariopsis brevicaulis, Fusarium solani, F. oxysporum, F. verticillioides, Candida albicans and C. tropicalis were used after confirmation of their specificity. Results, Forty-seven cases (94%) were positive for fungal DNA, among which dermatophytes were detected in 39 cases (83·0%): T. rubrum in 35 cases (74·5%) and T. mentagrophytes in eight cases (17·0%). Surprisingly, nondermatophytes were detected in 18 cases (38·3%), both dermatophytes and nondermatophytes in 10 cases (21·3%) and nondermatophytes alone in eight cases (17·0%). Aspergillus spp. alone was observed in five cases (10·6%). Conclusions, This study indicates that most of the affected nail plates of patients with onychomycosis were positive for specific fungal DNAs, and suggests that nondermatophytes detected at high rates may be involved in the pathogenesis of onychomycosis. [source] Detection of epidermodysplasia verruciformis-associated human papillomavirus DNA in nongenital seborrhoeic keratosisBRITISH JOURNAL OF DERMATOLOGY, Issue 5 2004Y-H. Li Summary Background, DNA of epidermodysplasia verruciformis (EV)-associated human papillomaviruses (HPVs) has been widely detected in lesions of malignant skin tumours, benign tumours and other proliferative diseases of epithelial origin. Objectives, To investigate the presence of EV-associated HPV DNA in nongenital seborrhoeic keratosis (SK) and to elucidate the prevalence of distinct HPV genotypes. Methods, We investigated HPV DNA in 55 nongenital SK biopsies, which were compared with 48 normal skin biopsies (healthy controls) using a nested polymerase chain reaction (PCR) using consensus primers CP65/CP70 and CP66/CP69. The positive PCR products were retracted and used to prepare recombination clones with T-vector. Distinct clones were analysed with endonucleases, and HPV genotypes were identified by direct sequencing. Results, EV-associated HPV DNA was detected in 42 of 55 (76%) nongenital SK biopsies vs. only 13 of 48 (27%) healthy controls (,2 = 22·087; P < 0·005). The prevalence was higher in patients with more than five lesions than in those with only one lesion (P < 0·05). Ten distinct HPV genotypes were detected in the nongenital SK biopsies: HPV 20, 23, 5, renal transplant recipient (RTR) X7, HPV 17, 37, 17b, RTRX4, RTRX4b and strain SK3. HPV 20 was found in 26 of 42 (62%) positive specimens, followed by HPV 23 (11 of 42, 26%) and HPV 5 (six of 42, 14%). Existence of multiple HPV genotypes was observed in 12 of 42 (29%) positive specimens. In healthy controls, five genotypes of EV-associated HPV (HPV 20, 23, 5, 17 and RTRX4) were detected, with the same predominant genotype of HPV 20 (five of 13, 38%). Several distinct HPV genotypes were found to coexist in four of 13 (31%) positive specimens. Conclusions, This study provides some evidence that EV-associated HPVs might play a part in the pathogenesis of nongenital SK. [source] Herpesviral DNA in brain tissue from patients with temporal lobe epilepsyACTA NEUROLOGICA SCANDINAVICA, Issue 3 2004O. Eeg-Olofsson Objectives , Presence of DNA from six herpesviruses were examined in brain tissue from patients operated for temporal lobe epilepsy. Material and methods , A total of 19 Canadian patients (I) with a median age of 22 years, 17 Swedish patients (II) with a median age of 14 years and a reference group comprising 12 individuals were studied. Presence of herpesviral DNA was detected by nested polymerase chain reaction. Results , Of three children with Rasmussen's encephalitis, Cytomegalovirus (CMV) DNA was found in two, and human herpesvirus type 6 DNA in two. In six children with ganglioglioma, Epstein,Barr virus (EBV) was detected in four. CMV DNA was found significantly more in group I compared with II, while the reverse occurred with EBV DNA. Malformations of cortical development were found significantly more in group II compared with I. Conclusion , Detection of DNA from some herpesviruses in epileptic brain tissue may possibly be associated with distinct clinical conditions, but factors such as age and malformations of cortical development should also be considered. [source] Detection and molecular characterization of foamy viruses in Central African chimpanzees of the Pan troglodytes troglodytes and Pan troglodytes vellerosus subspeciesJOURNAL OF MEDICAL PRIMATOLOGY, Issue 2 2006Sara Calattini Abstract Background, Foamy viruses are exogenous retroviruses that are highly endemic in non-human primates (NHPs). Recent studies, mainly performed in North America, indicated frequent simian foamy virus (SFV) infection in persons occupationally exposed to NHPs. This zoonotic infection was demonstrated mainly after bites by chimpanzees [Pan troglodytes (P. t.)] of the West African P. t. verus subspecies in primatology centers or zoos in the USA. Methods, We studied 32 chimpanzees from the Central African subspecies P. t. troglodytes and P. t. vellerosus, originating from Cameroon (29 cases) or Gabon (3 cases). We screened first plasma or sera of the animals with a Western blot detecting the SFVs Gag doublet proteins. Then, we performed two nested polymerase chain reactions (PCRs) amplifying a fragment of the integrase and LTR regions and, finally, we made phylogenetical analyses on the sequences obtained from the integrase PCR products. Results, By serological and/or molecular assays, we detected foamy viruses (FVs) infection in 14 chimpanzees. Sequence comparison and phylogenetic analyses of a 425 bp fragment of the integrase gene obtained for 10 of the 14 positive apes, demonstrated a wide diversity of new FVs strains that belong phylogenetically either to the P. t. troglodytes or P. t. vellerosus foamy viral clade. Conclusions, This study shows that chimpanzees living in these areas of Central Africa are infected by several specific foamy viruses. This raises, in such regions, the potential risk of a human retroviral infection of zoonotic origin linked to chimpanzees contacts, as already exemplified for STLV-1 and SIV infections. [source] | ||||||||||||||||||||||||||||