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Primer Sets (primer + set)
Kinds of Primer Sets Selected AbstractsThe Application of Miniplex Primer Sets in the Analysis of Degraded DNA from Human Skeletal Remains§JOURNAL OF FORENSIC SCIENCES, Issue 2 2006Kerry L. Opel M.A. ABSTRACT: A new set of multiplexed PCR primers has been applied to the analysis of human skeletal remains to determine their efficacy in analyzing degraded DNA. These primer sets, known as Miniplexes, produce shorter amplicons (50,280 base pairs (bp)) than standard short tandem repeat (STR) kits, but still utilize the 13 CODIS STR loci, providing results that are searchable on national DNA databases. In this study, a set of 31 different human remains were exposed to a variety of environmental conditions, extracted, and amplified with commercial and Miniplex DNA typing kits. The amplification efficiency of the Miniplex sets was then compared with the Promega PowerPlex® 16 system. Sixty-four percent of the samples generated full profiles when amplified with the Miniplexes, while only 16% of the samples generated full profiles with the Powerplex® 16 kit. Complete profiles were obtained for 11 of the 12 Miniplex loci with amplicon sizes less than 200 bp. These data suggest smaller PCR amplicons may provide a useful alternative to mitochondrial DNA for anthropological and forensic analysis of degraded DNA from human skeletal remains. [source] Novel classical MHC class I alleles identified in horses by sequencing clones of reverse transcription-PCR productsINTERNATIONAL JOURNAL OF IMMUNOGENETICS, Issue 6 2003C. Chung Summary Improved typing of horse classical MHC class I is required to more accurately define these molecules and to extend the number identified further than current serological assays. Defining classical MHC class I alleleic polymorphism is important in evaluating cytotoxic T lymphocyte (CTL) responses in horses. In this study, horse classical MHC class I genes were analyzed based on reverse transcription (RT)-PCR amplification of sequences encoding the polymorphic peptide binding region and the more conserved alpha 3, transmembrane and cytoplasmic regions followed by cloning and sequencing. Primer sets included a horse classical MHC class I-specific reverse primer and a forward primer conserved in all known horse MHC class I genes. Sequencing at least 25 clones containing MHC class I sequences from each of 13 horses identified 25 novel sequences and three others which had been described. Of these, nine alleles were identified from different horses or different RT-PCR and 19 putative alleles were identified in multiple clones from the same RT-PCR. The primer pairs did not amplify putative non-classical MHC class I genes as only classical MHC class I and related pseudogenes were found in 462 clones. This method also identified classical MHC class I alleles shared between horses by descent, and defined differences in alleles between horses varying in equine leukocyte antigen (ELA)-A haplotype as determined by serology. However, horses sharing ELA-A haplotypes defined by serotyping did not always share cDNA sequences, suggesting subhaplotypic variations within serologically defined ELA-A haplotypes. The 13 horses in this study had two to five classical MHC class I sequences, indicating that multiple loci code for these genes. Sequencing clones from RT-PCR with classical MHC class I-specific primers should be useful for selection of haplotype matched and mismatched horses for CTL studies, and provides sequence information needed to develop easier and more discriminating typing procedures. [source] Real-time primer design for DNA chipsCONCURRENCY AND COMPUTATION: PRACTICE & EXPERIENCE, Issue 9 2004H. Simmler Abstract The design of PCR or DNA chip experiments is a time-consuming process where bioinformatics is extensively used. The selection of the primers, which are immobilized on the DNA chip, requires a complex algorithm. Based on several parameters an optimized set of primers is automatically determined for a given gene sequence. This paper describes a parallel architecture which performs the optimization of the primer selection on a hardware accelerator. In contrast to the pure software approach, the parallel architecture gains a speedup of factor 500 using a PCI-based hardware accelerator. This approach allows an optimization of a specified primer set in real time. Copyright © 2004 John Wiley & Sons, Ltd. [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] The potential impact of human papillomavirus vaccination in contemporary cytologically screened populations may be underestimated: An observational retrospective analysis of invasive cervical cancersINTERNATIONAL JOURNAL OF CANCER, Issue 10 2009Ned Powell Abstract The aim of this study was to determine the proportion of invasive cervical cancers attributable to human papillomavirus (HPV) types 16 and 18 in a contemporary, cytologically well-screened UK population. This was achieved in a retrospective observational analysis by HPV typing 453 archival invasive cervical cancers diagnosed between January 1, 2000 and September 1, 2006. Pathological material was collected from 9 hospitals across Wales (UK), and HPV typing and pathology review was conducted at a central laboratory. Genotyping for high-risk HPV DNA was performed by PCR-enzyme immunoassay using the GP5+/6+ primer set. DNA was successfully extracted from 297 cases. Two hundred and eighty cases were included in the final analysis. The proportion of cases which had only HPV 16 and/or 18 was 219 of 280 (78.2%, 95% CI = 73.0,82.7); the proportion of cases which had HPV 16 or 18 and another HPV type was 230 of 280 (82.1%, 95% CI = 77.2,86.2). The proportion of cervical cancers associated with infection with HPV types 16 and 18 has previously been estimated at around 70%. The appropriate figure for a cytologically well-screened UK population appears to be approximately 80%. Hence, the potential impact of the current vaccination programme may be underestimated. © 2009 UICC [source] Development and comparison of SYBR Green quantitative real-time PCR assays for detection and enumeration of sulfate-reducing bacteria in stored swine manureJOURNAL OF APPLIED MICROBIOLOGY, Issue 6 2008C. Spence Abstract Aims:, To develop and evaluate primer sets targeted to the dissimilatory sulfite reductase gene (dsrA) for use in quantitative real-time PCR detection of sulfate-reducing bacteria (SRB) in stored swine manure. Methods and Results:, Degenerate primer sets were developed to detect SRB in stored swine manure. These were compared with a previously reported primer set, DSR1F+ and DSR-R, for their coverage and ability to detect SRB communities in stored swine manure. Sequenced clones were most similar to Desulfovibrio sp. and Desulfobulbus sp., and these SRB populations differed within different manure ecosystems. Sulfur content of swine diets was shown to affect the population of Desulfobulbus -like Group 1 SRB in manure. Conclusions:, The newly developed assays were able to enumerate and discern different groups of SRB, and suggest a richly diverse and as yet undescribed population of SRB in swine manure. Significance and Impact of the Study:, The PCR assays described here provide improved and efficient molecular tools for quantitative detection of SRB populations. This is the first study to show population shifts of SRB in swine manure, which are a result of either the effects of swine diets or the maturity of the manure ecosystem. [source] Quantitative analysis of human enteric adenoviruses in aquatic environmentsJOURNAL OF APPLIED MICROBIOLOGY, Issue 6 2007E. Haramoto Abstract Aims:, The aim of this study was to determine human adenoviruses (HuAdVs) in aquatic environments by real-time polymerase chain reaction (PCR). Methods and Results:, In order to describe the ratio of enteric serotypes to the total HuAdVs, the primer set specific for the enteric serotypes 40 and 41 was used in parallel with the universal primer set for all 51 serotypes of HuAdVs. The enteric serotypes of HuAdVs were detected at the concentration of 7·3,1500 PCR-detection units (PDU) per ml in raw sewage (n = 17), 0·00060,4·1 PDU ml,1 in secondary-treated sewage before chlorination (n = 17), 0·0018,7·0 PDU ml,1 in river water (n = 36), and 0·032,6·1 PDU ml,1 in seawater (n = 18). The concentration of HuAdVs, determined by the universal primer set, was equivalent to that of enteric serotypes in almost all the samples tested. Conclusions:, Enteric serotypes were predominant among all serotypes of HuAdVs in the aquatic environments. Significance and Impact of the Study:, The abundance of enteric serotypes of HuAdVs should be more emphasized than other serotypes in order to assess the risk of their infection via water. [source] Detection of spring viraemia of carp virus (SVCV) by loop-mediated isothermal amplification (LAMP) in koi carp, Cyprinus carpio LJOURNAL OF FISH DISEASES, Issue 4 2008R B Shivappa Abstract Spring viraemia of carp virus (SVCV) is a rhabdovirus associated with systemic illness and mortality in cyprinids. Several diagnostic tests are available for detection of SVCV. However, most of these tests are time consuming and are not well adapted for field-based diagnostics. In this study, a diagnostic tool for SVCV detection based on reverse transcription loop-mediated isothermal amplification (RT-LAMP) has been developed. Based on the nucleotide sequence of the glycoprotein (G) gene of SVCV North Carolina (NC) isolate, four sets (each set containing two outer and two inner) of primers were designed. Temperature and time conditions were optimized to 65 °C and 60 min, respectively, for LAMP and RT-LAMP using one primer set. In vitro specificity was evaluated using four different strains of fish rhabdoviruses and RT-LAMP was found to be specific to SVCV. Serial dilutions of SVCV NC isolate was used to evaluate the in vitro sensitivity of RT-LAMP. Sensitivity of the assays was similar to RT-PCR and detected SVCV even at the lowest dilution of 101 TCID50 mL,1. The ability of RT-LAMP to detect SVCV from infected carp was also tested and the assay detected SVCV from all infected fish. The isothermal temperature requirements, high specificity and sensitivity, and short incubation time of the RT-LAMP assay make it an excellent choice as a field diagnostic test for SVCV. [source] Comparative challenge model of Flavobacterium columnare using abraded and unabraded channel catfish, Ictalurus punctatus (Rafinesque)JOURNAL OF FISH DISEASES, Issue 8 2003J A Bader Abstract The early entry of the fish pathogen Flavobacterium columnare and enhancement by abrasion was studied in channel catfish, Ictalurus punctatus (Rafinesque), using the polymerase chain reaction and a species-specific primer set for a bacterial 16S rRNA gene product. Evaluations were conducted following an abrasion bath immersion challenge with F. columnare. Abrasion, a practice which has historically been used prior to bacterial challenge, had significant effects on the early entry of the pathogen and on cumulative percent survival (CPS). The FvpF1,FvpR1 primer set was useful in detecting the early entry of F. columnare in mucus, skin, gill, blood, liver and trunk kidney tissues in both abraded and unabraded fish following immersion challenge at 29 ± 2 °C. Bacteria were detected earlier in all tissues in abraded fish, except in the trunk kidney. These differences were not significant, except in the case of blood. Mucus, skin and gill tissues were positive for F. columnare earliest regardless of treatment (after 5 min in abraded fish and after 15 min in unabraded fish). CPS following challenge with F. columnare was significantly affected by abrasion, which supports the use of abrasion for the F. columnare challenge model for channel catfish. [source] Association between human papillomavirus infection and laryngeal squamous cell carcinomaJOURNAL OF MEDICAL VIROLOGY, Issue 6 2010Kamal Morshed Abstract The aim of this study was to compare the prevalence of human papillomavirus (HPV) infection in laryngeal squamous cell carcinoma using two methods: PCR-DNA enzyme immunoassay (PCR/DEIA) and immunohistochemistry (IHC) for detection of HPV in specimens of laryngeal squamous cell carcinoma and to correlate the presence of HPV with the epidemiological and clinicopathological features of recurrence and survival. HPV DNA was amplified from 93 paraffin-embedded laryngeal squamous cell carcinoma tissue specimens by the short PCR fragment (SPF 10) primer set using PCR/DNA method. HPV detection using monoclonal anti-human papilloma virus antibodies Clone K1H8 for IHC reaction was performed on 130 specimens. HPV was identified in 35.5% of patients with laryngeal squamous cell carcinoma using PCR/DEIA and 27.7% using IHC. There was no statistically significant association between the presence of HPV and the epidemiological and clinicopathological features and recurrence. There was no statistically significant association between the presence of HPV and overall survival nor disease specific survival. Statistically significant correlation between HPV detection using PCR/DEIA technique and IHC technique was found. The presence of HPV infection in 27.7% and 38.9% of the patients suggests a possible role in the etiology of laryngeal squamous cell carcinoma. The SPF10 PCR/DEIA technique is the most accurate method for detection of HPV in laryngeal squamous cell carcinoma. J. Med. Virol. 82:1017,1023, 2010. © 2010 Wiley-Liss, Inc. [source] Evaluation and application of reverse transcription loop-mediated isothermal amplification for detection of noroviruses,JOURNAL OF MEDICAL VIROLOGY, Issue 3 2007Tomoko Yoda Abstract A one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the detection of norovirus (NV) was developed. In order to design primer sets for the detection of a wide range of NVs, NVs were categorized into three groups, that is, genogroup I (GI), prevalent GII, and minor GII; three sets of primers were developed for each group. Clinical specimens of patients suffering from enteric RNA viruses, such as NV, group A and C rotavirus, and sapovirus were examined using these primer sets. Various genotypes of NVs were detected in clinical specimens from patients infected with NV where no false positive reaction was observed with other enteric RNA viruses. Additionally, 88 samples of acute gastroenteritis outbreaks were analyzed by an RT-LAMP assay and compared with the results of routine RT-PCR. The results of the RT-LAMP assay corresponded well to that of RT-PCR. These findings suggest the practical application of the RT-LAMP assay for the detection of NVs in clinical specimens. Consequently, the RT-LAMP system and conventional detection kits (NVGI and NVGII detection kits; Eiken Chemical Co., Ltd., Japan) were compared. The detection rate of the prevalent and minor GII primer sets was similar to that of the conventional NVGII kit, while the detection rate of the GI primer set is different because it can detect several genotypes better than the conventional NVGI kit. This is an initial report that the RT-LAMP system is able to detect NVs in clinical specimens within a wide range. J. Med. Virol. 79:326,334, 2007. © 2007 Wiley-Liss, Inc. [source] Gene Expression Profiles of Intracellular and Membrane Progesterone Receptor Isoforms in the Mediobasal Hypothalamus During Pro-OestrusJOURNAL OF NEUROENDOCRINOLOGY, Issue 12 2009B. Liu Progesterone action is mediated by its binding to specific receptors. Two progesterone receptor (PR) isoforms (PRA and PRB), three membrane progesterone receptor (mPR) subtypes (mPR,, mPR, and mPR,) and at least one progesterone membrane-binding protein [PR membrane component 1 (PRmc1)] have been identified in reproductive tissues and brain of various species. In the present study, we examined gene expression patterns for PR isoforms, mPR subtypes and PRmc1 in the rat mediobasal hypothalamus (MBH) during pro-oestrus. The mRNA level for each receptor subtype was quantified by a real-time reverse transcriptase-polymerase chain reaction (RT-PCR) at the time points: 13.00 h on dioestrous day 2; 09.00, 13.00, 17.00 and 22.00 h on pro-oestrus; and 13.00 h on oestrus. For PR, one primer set amplified PRA+PRB, whereas a second primer set amplified PRB. As expected, PRA+PRB mRNA expression was greater than PRB in MBH tissue. PRB mRNA levels increased throughout the day on pro-oestrus, with the highest levels being observed at 17.00 h. PRB mRNA levels in the MBH were increased by 2.4- and 3.0-fold at 13.00 and 17.00 h, respectively, on pro-oestrus compared to 13.00 h on dioestrous day 2. There were differential mRNA expression levels for mPRs and PRmc1 in the MBH, with the highest expression for PRmc1 and the lowest for mPR,. The mPR, mRNA contents at 13.00 and 17.00 h on pro-oestrus were increased by 1.5-fold compared to that at 13.00 h on dioestrous day 2. The mPR, mRNA levels at 13.00 and 17.00 h on pro-oestrus were 2.5- and 2.4-fold higher compared to that at 13.00 h on dioestrous day 2, respectively. PRA+PRB, mPR, and PRmc1 mRNA levels did not vary on pro-oestrus. These findings suggest that the higher expression of PRB, mPR, and mPR, in the MBH on pro-oestrous afternoon may influence both genomic and nongenomic mechanisms of progesterone action during the critical pre-ovulatory period. [source] OLIGONUCLEOTIDE PRIMERS FOR THE DETECTION OF BIOLUMINESCENT DINOFLAGELLATES REVEAL NOVEL LUCIFERASE SEQUENCES AND INFORMATION ON THE MOLECULAR EVOLUTION OF THIS GENE,JOURNAL OF PHYCOLOGY, Issue 2 2008Andrea Baker Bioluminescence is reported in members of 18 dinoflagellate genera. Species of dinoflagellates are known to have different bioluminescent signatures, making it difficult to assess the presence of particular species in the water column using optical tools, particularly when bioluminescent populations are in nonbloom conditions. A "universal" oligonucleotide primer set, along with species and genus-specific primers specific to the luciferase gene were developed for the detection of bioluminescent dinoflagellates. These primers amplified luciferase sequences from bioluminescent dinoflagellate cultures and from environmental samples containing bioluminescent dinoflagellate populations. Novel luciferase sequences were obtained for strains of Alexandrium cf. catenella (Whedon et Kof.) Balech and Alexandrium fundyense Balech, and also from a strain of Gonyaulax spinifera (Clap. et Whitting) Diesing, which produces bioluminescence undetectable to the naked eye. The phylogeny of partial luciferase sequences revealed five significant clades of the dinoflagellate luciferase gene, suggesting divergence among some species and providing clues on their molecular evolution. We propose that the primers developed in this study will allow further detection of low-light-emitting bioluminescent dinoflagellate species and will have applications as robust indicators of dinoflagellate bioluminescence in natural water samples. [source] Aromatic hydrocarbon degradation genes from chronically polluted Subantarctic marine sedimentsLETTERS IN APPLIED MICROBIOLOGY, Issue 5 2009M.S. Marcos Abstract Aim:, The goal of this study was to identify functional targets to detect polycyclic aromatic hydrocarbon (PAH)-degrading bacterial populations in cold marine ecosystems. Methods and Results:, We designed a degenerate primer set targeting genes encoding the , subunit of PAH-dioxygenases from Gram-positive bacteria. This primer set was used to amplify gene fragments from metagenomic DNA isolated from Subantarctic marine sediments (Ushuaia Bay, Argentina). These gene fragments were cloned and sequenced. We identified 14 distinct groups of genes, most of them showing significant relatedness with dioxygenases from Gram-positive bacteria of the genera Rhodococcus, Mycobacterium, Nocardioides, Terrabacter and Bacillus. The level of identity with these genes, however, was low to moderate (33,62% at the amino acid level). Conclusion:, These results indicate the presence of a high diversity of hitherto unidentified dioxygenase genes in this cold polluted environment. Significance and Impact of the Study:, Subantarctic marine ecosystems are particularly vulnerable to hydrocarbon pollution, and the development of environmental restoration strategies for these environments is pressing. The information obtained in this work will be the starting point for the design of quantitative molecular tools to analyse the abundance and dynamics of these aromatic hydrocarbon-degrading bacterial populations in the marine environment. [source] Identification of Lactobacillus crispatus by polymerase chain reaction targeting S-layer protein geneLETTERS IN APPLIED MICROBIOLOGY, Issue 1 2002M. Horie Aims: ,This study aimed to develop a polymerase chain reaction (PCR) method to identify Lactobacillus crispatus . Methods and Results: ,A primer set (CbsA2F,CbsA2R) for amplifying conserved regions of S-layer genes was designed to identify Lact. crispatus and the specificity of this set was compared with that of another primer set (Cri 16SI,Cri 16SII) which has been reported as a species-specific primer set targeting the 16S rRNA gene. Among species in the Lact. acidophilus A1,A4 groups, when KOD polymerase was used for amplification, the primer set CbsA2F,CbsA2R gave PCR products with Lact. crispatus strains only. However, when Taq polymerase was used, this primer set gave products with one Lact. amylovorus strain as well as with Lact. crispatus strains. The primer set Cri 16SI,Cri 16SII gave PCR products with Lact. crispatus strains and two Lact. acidophilus strains, regardless of whether the polymerase used was KOD or Taq . Conclusions: ,A PCR targeting the S-layer gene and amplified with KOD polymerase can identify Lact. crispatus accurately and rapidly. Significance and Impact of the Study: ,To the authors' knowledge, this is the first paper to provide a PCR method for the specific identification of Lact. crispatus . [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] A set of 16 consensus primer pairs amplifying the complete mitochondrial genomes of orange-spotted grouper (Epinephelus coioides) and Hong Kong grouper (Epinephelus akaara)MOLECULAR ECOLOGY RESOURCES, Issue 6 2009XUAN ZHUANG Abstract Groupers are of considerable economic value; however, their classification and evolutionary relationships have long been hindered by the overwhelming number of species and lack of morphological specializations. Mitochondrial genome is a source of original markers that are potentially useful in the study of phylogeny and population genetics of groupers. We describe a set of 16 new primer pairs that allow PCR amplification of the entire mitochondrial genomes of orange-spotted grouper and Hong Kong grouper. This primer set has been defined for consensus over eight other grouper species, facilitating further studies on the molecular evolution and population genetics of groupers. [source] Identification of the endangered small red brocket deer (Mazama bororo) using noninvasive genetic techniques (Mammalia; Cervidae)MOLECULAR ECOLOGY RESOURCES, Issue 3 2009SUSANA GONZÁLEZ Abstract The small red brocket deer Mazama bororo is one of the most endangered deer in the Neotropics. The great morphological similarities with three other sympatric brocket deer species, coupled with the fact that they inhabit densely forested habitats complicate detection and prevent the use of traditional methodologies for accurate identification of species. The ability to determine the presence of this endangered species in an area is crucial for estimating its distribution range, and is critical for establishing conservation management strategies. Here we describe a fast and reliable noninvasive genetic method for species identification of Mazama species from faeces. We designed a primer set that amplifies a short 224-bp fragment of the cytochrome b and demonstrate its effectiveness in successful amplification of DNA isolated from both tissue and faecal samples. This fragment contains a BSTNI/ECORII digestion site that is unique to the endangered M. bororo. The digested polymerase chain reaction products yielded a 160-bp fragment that is clearly visible in a 2% agarose gel. Two other diagnostic sites were identified to differentiate the other three sympatric species, SspI (M. gouazoubira) and AflIII (M. americana, and M. nana). [source] Prevalence and molecular diversity of Archaea in subgingival pockets of periodontitis patientsMOLECULAR ORAL MICROBIOLOGY, Issue 4 2009C. L. Li Introduction:, The aim of this study was to investigate the prevalence and molecular diversity of Archaea in the subgingival crevices of patients with chronic periodontitis. Methods:, Subgingival plaque was collected from 41 patients with chronic periodontitis and 15 healthy subjects. The prevalence of Archaea in those plaque samples was tested by polymerase chain reaction with two broad-range archaeal primer sets. Amplicons from eight Archaea -positive plaque samples were cloned and sequenced for molecular diversity analysis using one of these two primer sets and a novel third primer set. Results:,Archaea were detected in the subgingival plaque of patients with chronic periodontitis at a prevalence of 70.7,73.2%, but were not detected in healthy subjects. Using one primer set, all sequences of the archaeal amplicons were identified as Methanobrevibacter oralis -like species. With another primer set, the amplicons were also found to be identical to the uncultured M. oralis -like species except one phylotype was found to belong to the class Thermoplasmata. Conclusion:,Archaea might be correlated with periodontal diseases. The diversity of Archaea associated with periodontitis was limited. Almost all sequenced amplicons fell into the genus Methanobrevibacter of the Euryarcheota phylum. M. oralis -like species was the predominant but non-exclusive archaeon in the subgingival dental plaque of patients with periodontitis. [source] Diversity and detection of Korean Erwinia pyrifoliae strains as determined by plasmid profiling, phylogenetic analysis and PCRPLANT PATHOLOGY, Issue 6 2007R. Shrestha Twenty-five strains of Erwinia pyrifoliae were investigated for their plasmid profiles and genetic relatedness. Four types of plasmid profile were observed for the first time, suggesting intraspecific plasmid profile diversity in E. pyrifoliae. Moreover, BOX-PCR and phylogenetic analysis based on the 16S-23S intergenic transcribed spacer (ITS) region showed genetic variations among E. pyrifoliae strains, although all strains were clustered in one group and separated from E. amylovora. On the other hand, ERIC-PCR and phylogenetic analysis based on partial groEL gene sequences revealed close genetic relatedness among the strains. Amplification with EpSPF and EpSPR primers of a fragment of approximately 0·65 kb from the genomic DNA of all E. pyrifoliae strains, but not from E. amylovora strains, suggested that this primer set is useful for identification of this pathogen. [source] An evaluation of PCR primer sets used for detection of Propionibacterium acnes in prostate tissue samplesTHE PROSTATE, Issue 14 2008Karen S. Sfanos Abstract BACKGROUND Multiple studies have now shown that Propionibacterium acnes can be cultured from post-prostatectomy derived prostate tissue samples. In contrast, both universal eubacterial 16S rDNA PCR and P. acnes -specific 16S rDNA PCR have failed to detect this organism at a frequency similar to that of bacterial culture. A potential explanation for this discrepancy, proposed by Cohen et al., involves mismatches in 16S rDNA primer sets used for bacterial detection. METHODS The sensitivity of both a previously published P. acnes -specific primer set containing a potential mismatch and a new primer set with no mismatches was determined. Both primer sets were used to interrogate two sets of DNA samples derived from post-prostatectomy prostate tissues that differed in the level of sterile precautions maintained during tissue collection. RESULTS The number of P. acnes positive samples was associated with the sterility of the sample collection process. In all instances, positive samples were determined to reflect low cell numbers (<10 CFU). CONCLUSIONS Although the results of previous studies have shown that P. acnes is not the only organism potentially present in the prostates of prostate cancer patients, mismatches in PCR primer sets may have also influenced the sensitivity of P. acnes detection. When using PCR in determining the presence of P. acnes in the human prostate, care should be taken to establish the potential influence of exogenous contamination and, due to the sensitivity of the assay, samples exposed to the urethra during the collection process (prostatic secretions, TURP specimens) should not be used. Prostate 68: 1492,1495, 2008. © 2008 Wiley-Liss, Inc. [source] Temporal arteritis and Chlamydia pneumoniae: Failure to detect the organism by polymerase chain reaction in ninety cases and ninety controlsARTHRITIS & RHEUMATISM, Issue 4 2002Michael J. Regan Objective To examine the reported correlation between the presence of Chlamydia pneumoniae in temporal artery biopsy specimens and the diagnosis of temporal arteritis (TA). Methods Among 90 possible cases of TA identified at our institution between 1968 and 2000, 79 of the positive biopsy specimens (88%) demonstrated giant cells and the other 11 cases (12%) had other histopathologic features compatible with TA; by chart review, all 90 patients were confirmed to have met the American College of Rheumatology classification criteria for TA. Controls had negative temporal artery biopsy specimens during the same 32-year time period and their postbiopsy disease courses were not compatible with TA. Controls were matched with each case by sex, year of biopsy, and age within 10 years. The biopsy specimens from all cases and controls were reevaluated and readings were confirmed in a masked manner by an experienced eye pathologist. Polymerase chain reaction (PCR) analyses for C pneumoniae were performed on the 180 samples using 2 different sets of PCR primers (which target 2 different genes). A primer set targeting the ompA gene (CP1-CP2/CPC-CPD) was used to perform a nested PCR, followed by confirmation of the findings with primers targeting the 16S ribosomal RNA (rRNA) gene (Cpn90/Cpn91) in a touchdown-enzyme time-release PCR. We used positive and negative controls, as well as controls made from infected and noninfected HEp-2 cells, suspended in a formalin-fixed, paraffin-embedded matrix. Results Seventy-six percent of the 180 cases and controls were women. The mean age of the cases was 72.0 years (range 53,90), and that of the controls was 70.4 years (range 51,86). Eighty percent of the control samples were obtained by temporal artery biopsy performed within 1 year of the biopsies performed on the matched cases. Using the CP1-CP2/CPC-CPD primer set, only 1 TA case sample (1% of all case samples) was positive for the ompA gene. One control sample was also positive using these primers. With the Cpn90/Cpn91 primers, none of the cases and none of the controls were positive for the 16S rRNA gene. Conclusion The results of this study using sensitive and specific PCR analyses do not support a role for C pneumoniae in the pathogenesis of TA. [source] Real-time Polymerase Chain Reaction to Follow the Response of Muscle to TrainingARTIFICIAL ORGANS, Issue 8 2008Lauren M. Moore Abstract:, The adaptive response of muscle to changes in activity or loading can take many weeks. Changes in the levels of RNA within a muscle fiber can give an early indication of the nature of the response of that fiber to changes in activity or loading. We have designed a new primer set for quantitative polymerase chain reaction (PCR) that will allow us to follow these early transcriptional changes in rat muscle, and have shown that analysis can be performed by standard techniques on as little as 5 mg of muscle, an amount that can be obtained by needle biopsy. [source] Detection of human papillomavirus DNA in squamous cell carcinoma of the esophagus by auto-nested PCRDISEASES OF THE ESOPHAGUS, Issue 2 2006A. P. Souto Damin SUMMARY., The aim of the present study was to investigate the presence of human papillomavirus (HPV) in surgical specimens of esophageal squamous cell carcinoma. One hundred and sixty-five paraffin-embedded specimens of esophageal carcinoma were analyzed through high-sensitivity auto-nested polymerase chain reaction (PCR) using the consensus GP5+/GP6+ primer. Twenty-six specimens of esophageal mucosa without malignant disease were also studied as a control group. Two different specific primer sets targeting the E6 region of the HPVs 16 and 18 were used for typing. Direct DNA sequence analysis was conducted to confirm positive PCR results. HPV DNA was detected in 26 esophageal carcinomas (15.75%), but in none of the benign esophageal specimens (P < 0.05). Out of the 26 positive cases, 24 were HPV-16 and one was HPV-18. One tumor contained both HPV-16 and -18 DNA. Positive PCR results were confirmed by the amplified viral sequences. Our findings suggest that the presence of either HPV-16 or -18 might be related to development of the malignant phenotype in the esophagus. [source] Polymorphic microsatellite loci for the swarm-founding wasp Polybia paulista (Hymenoptera: Vespidae)ENTOMOLOGICAL SCIENCE, Issue 1 2005Kazuyuki KUDÔ Abstract A polymorphic microsatellite locus was isolated and characterized from Polybia paulista, one of the most common polygynic, swarm-founding social wasps in Brazil. Three other microsatellite loci for which the primer sets were originally developed in independent-founding paper wasps also showed polymorphism in the size of amplification products in P. paulista. [source] Transcript and activity levels of different Pleurotus ostreatus peroxidases are differentially affected by Mn2+ENVIRONMENTAL MICROBIOLOGY, Issue 5 2001Roni Cohen The white-rot fungus Pleurotus ostreatus produces both manganese-dependent peroxidase (MnP) and versatile peroxidase (VP) in non-manganese-amended peptone medium (PM). We studied the effect of Mn2+ supplementation on MnPs and VPs in P. ostreatus by analysing the enzymatic and transcript abundance profiles of the peroxidases, as well as the lignin mineralization rate. The fungus was grown in PM under solid-state conditions using perlite as an inert solid support. Mn2+ amendment resulted in a 1.7-fold increase in [14C]-lignin mineralization relative to unamended medium. Anion-exchange chromatography was used to resolve the fungal peroxidase's enzymatic activity profile. Five peaks (P1,P5) of VP and one peak (P6) of MnP activity were detected in unamended medium. In Mn2+ -amended medium, a reduction in the activity of the VPs was observed. On the other hand, a sharp increase in the MnP activity level of peak P6 was detected. The P6 isoenzyme was purified and showed manganese-dependent peroxidation of phenolic substrates. Internal sequence analysis of the purified enzyme revealed 100% identity with the deduced amino acid sequence of P. ostreatus MnP3 (GenBank AB016519). The effect of Mn2+ on the relative abundance of gene transcripts of three VPs and one MnP from P. ostreatus was monitored using reverse transcription,polymerase chain reaction (RT,PCR) with oligonucleotide primer sets synthesized on the basis of non-conserved sequences of the different peroxidases. The reduction in VP gene transcript abundance and the increase in mnp3 transcript level were collinear with the changes observed in the enzyme activity profiles. These results indicate that the activity of peroxidases is regulated at the transcriptional level. We suggest that the expression of MnP and VP may be differentially regulated by the presence of Mn2+. [source] Identification of potentially toxic environmental Microcystis by individual and multiple PCR amplification of specific microcystin synthetase gene regionsENVIRONMENTAL TOXICOLOGY, Issue 3 2005Youness Ouahid Abstract Reliable cyanotoxin monitoring in water reservoirs is difficult because of, among other reasons, unpredictable changes in cyanobacteria biomass, toxin production, and inadequate sampling frequency. Therefore, it would be useful to identify potentially microcystin-producing strains of cyanobacterial populations in field samples. With this aim, we developed a methodology to distinguish microcystin-producing from non-producing Microcystis strains by amplifying six characteristic segments of the microcystin synthetase mcy cluster, three corresponding to the nonribosomal peptide synthetase, genes mcyA, mcyB, and mcyC, and three to the polyketide synthase, genes mcyD, mcyE, and mcyG. For this purpose five new primer sets were designed and tested using purified DNA, cultured cells, and field colonies as DNA sources. Simultaneous amplification of several genes in multipex PCR reactions was performed in this study. The results obtained showed that: (i) the expected specific amplicons were obtained with all microcystin-producing strains but not with nonproducing strains; (ii) cells could be directly used as DNA templates, 2000 cells being a sufficient number in most cases; (iii) simultaneous amplification of several gene regions is feasible both with cultured cells and with field colonies. Our data support the idea that the presence of various mcy genes in Microcystis could be used as a criterion for ascribing potential toxigenicity to field strains, and the possibility of applying whole-cell assays for the simultaneous amplification of various genes may contribute significantly to simplifying toxigenicity testing. © 2005 Wiley Periodicals, Inc. Environ Toxicol 20: 235,242, 2005. [source] Exploring the diversity of bacterial communities in sediments of urban mangrove forestsFEMS MICROBIOLOGY ECOLOGY, Issue 1 2008Newton C. Marcial Gomes Abstract Municipal sewage, urban runoff and accidental oil spills are common sources of pollutants in urban mangrove forests and may have drastic effects on the microbial communities inhabiting the sediment. However, studies on microbial communities in the sediment of urban mangroves are largely lacking. In this study, we explored the diversity of bacterial communities in the sediment of three urban mangroves located in Guanabara Bay (Rio de Janeiro, Brazil). Analysis of sediment samples by means of denaturing gradient gel electrophoresis (DGGE) of 16S rRNA gene fragments suggested that the overall bacterial diversity was not significantly affected by the different levels of hydrocarbon pollution at each sampling site. However, DGGE and sequence analyses provided evidences that each mangrove sediment displayed a specific structure bacterial community. Although primer sets for Pseudomonas, alphaproteobacterial and actinobacterial groups also amplified ribotypes belonging to taxa not intended to be enriched, sequence analyses of dominant DGGE bands revealed ribotypes related to Alteromonadales, Burkholderiales, Pseudomonadales, Rhodobacterales and Rhodocyclales. Members of these groups were often shown to be involved in aerobic or anaerobic degradation of hydrocarbon pollutants. Many of these sequences were only detected in the sampling sites with high levels of anthropogenic inputs of hydrocarbons. Many dominant DGGE ribotypes showed low levels of sequence identity to known sequences, indicating a large untapped bacterial diversity in mangrove ecosystems. [source] Multilocus sequence typing confirms synonymy but highlights differences between Candida albicans and Candida stellatoideaFEMS YEAST RESEARCH, Issue 5 2008Mette D. Jacobsen Abstract We used multi-locus sequence typing (MLST) to investigate 35 yeast isolates representing the two genome-sequenced strains plus the type strain of Candida albicans, four isolates originally identified as Candida stellatoidea type I and 28 representing type strains of other species now regarded as synonymous with C. albicans. DNA from all 32 C. albicans synonyms readily formed PCR products with the C. albicans MLST primer sets. Their sequences placed all of them within the existing C. albicans clade structure, represented by 1516 isolates. One isolate, originally received as Mycotorula sinensis, was resistant to flucytosine, but no other unusual susceptibilities were found to polyene, azole or echinocandin antifungal agents. The four isolates of C. stellatoidea type I coclustered with two other sucrose-negative isolates, originally identified as examples of Candida africana, in a group of strains highly distinct from the majority of C. albicans. Our results not only confirm the synonymity of all the isolates with C. albicans but also confirm an obvious genotypic difference in the case of C. stellatoidea type I. [source] Evidence for cyanophages active against bloom-forming freshwater cyanobacteriaFRESHWATER BIOLOGY, Issue 6 2008LI DENG Summary 1. A total of 35 putative cyanophages able to infect non-axenic cultures of bloom-forming freshwater cyanobacteria in the genera Microcystis, Anabaena and Planktothrix were isolated from Lake Zurich (Switzerland) and lakes in the Cotswold Water Park (U.K.). Eleven lytic cyanophage isolates were isolated on Microcystis and 12 each on Anabaena and Planktothrix. Cyanophage isolation protocols varied when using these different cyanobacterial hosts. 2. The collection of putative cyanophage isolates encompassed a variety of morphotypes, including the first filamentous cyanophage from any environment and the second siphocyanophage reported from fresh water. 3. PCR primer sets for gp20, gp23 and MCP genes, which have been previously found to be conserved in other cyanophages, were used in an attempt to determine genetic diversity among the phage isolates. The failure to obtain specific amplification products from most isolates suggests that the cyanophages isolated in this study were different from those previously characterized from both marine and freshwater environments. 4. Some putative cyanophages within the collection of isolates proved to have a very broad host range and were able to infect Anabaena, Microcystis and Planktothrix. The ability to infect a wide range of host taxa extends the potential reproductive period for lytic propagation, and also has implications for the transfer of genetic information between deeply separated cyanobacterial lineages. [source] | |||||||||||||||||||||||||||||||||||||