Produced Polymerase Chain Reaction Products Research Articles

Molecular organization of 5S rDNA intergenic spacer in Gentiana pneumonanthe L. and G. punctata L.

Aim. The study was aimed at cloning and analysis of molecular organization of 5S rDNA intergenic spacer (IGS) in two Gentiana species of Ukrainian flora, G. pneumonanthe L. and G. punctata L. Methods. 5S rDNA IGS sequence was amplified using polymerase chain reaction (PCR) with a pair of primers specific for the gene coding region. The produced PCR products were fractionated by gel-electrophoresis, isolated, ligated into plasmid pUC18, cloned into E. coli, and then sequenced. Nucleotide sequences were aligned using the Muscle algorithm and analyzed in the Unipro UGENE software. Results. The intergenic spacer region of the 5S rRNA genes was cloned and sequenced for two Gentiana species of Ukrainian flora, G. pneumonanthe and G. punctata. Based on the analysis of the alignment of the IGS sequences of five Gentiana species from three sections, some features of molecular organization of IGS of 5S rRNA genes in the studied species were established. In particular, motifs typical for other angiosperm families were identified, such as conservative oligo-dT motif at the IGS 3'-end that served as a transcription termination site and AT-rich region preceding the coding region of 5S rRNA gene. However, in the region of transcription initiation, conservative GC-element in position -13 is changed to AC. Conclusions. The interspecific variation of molecular organization of 5S rDNA IGS was identified among Gentiana species that can be used to clarify the phylogenetic relationships between members of this genus.Keywords: Gentiana species, 5S rDNA intergenic spacer, molecular organization, phylogeny.

Infrared laser ablation sample transfer of tissue DNA for genomic analysis.

Infrared (IR) laser ablation was used to remove material from tissue sections mounted on microscope slides, with subsequent capture in a solvent-containing microcentrifuge tube. Experiments conducted with a 3200-bp double-stranded plasmid DNA template demonstrated IR-laser ablation transfer of intact DNA. The transfer efficiency and the molecular integrity of the captured DNA were evaluated using Sanger sequencing, gel electrophoresis, and fluorimetric analysis. The plasmid DNA was reproducibly transferred with an efficiency of 59±3% at laser fluences of between 10 and 20kJ/m2 at a wavelength of 3μm. IR laser ablation sample transfer was then used to ablate and capture DNA from 50-μm-thick rat brain and kidney tissue sections. DNA was extracted from the captured material using five commercial DNA extraction kits that employed significantly divergent methodologies, with all kits recovering sufficient DNA for successful amplification by polymerase chain reaction (PCR). Four sets of primers were employed, targeting one region of the CYP 11b2 gene (376bp) and three different regions of the Snn1g gene (298, 168, and 281bp). The PCR results were not consistently reliable when using unpurified ablation samples; however, after extraction, all samples produced PCR products of the expected size. This work expands the sampling capabilities of IR laser ablation, demonstrating that DNA can be isolated from tissue samples for genomic assays. Due to the small size of the ablation regions (1mm2), this technique will be useful for sampling discrete cell populations from tissue sections. Graphical abstract Infrared laser ablation transfer of intact DNA from a tissue section.

Highly Sensitive End-Point PCR and SYBR Green qPCR Detection of Phymatotrichopsis omnivora, Causal Fungus of Cotton Root Rot.

Phymatotrichopsis omnivora, the causal pathogen of cotton root rot, is a devastating ascomycete that affects numerous important dicotyledonous plants grown in the southwestern United States and northern Mexico. P. omnivora is notoriously difficult to isolate from infected plants; therefore methods for accurate and sensitive detection directly from symptomatic and asymptomatic plant samples are needed for disease diagnostics and pathogen identification. Primers were designed for P. omnivora based on consensus sequences of the nuclear ribosomal internal transcribed spacer (ITS) region of geographically representative isolates. Primers were compared against published P. omnivora sequences and validated against DNA from P. omnivora isolates and infected plant samples. The primer combinations amplified products from a range of P. omnivora isolates representative of known ITS haplotypes using standard end-point polymerase chain reaction (PCR) methodology. The assays detected P. omnivora from infected root samples of cotton (Gossypium hirsutum) and alfalfa (Medicago sativa). Healthy plants and other relevant root pathogens did not produce PCR products with the P. omnivora-specific primers. Primer pair PO2F/PO2R was the most sensitive in end-point PCR assays and is recommended for use for pathogen identification from mycelial tissue and infected plant materials when quantitative PCR (qPCR) is not available. Primer pair PO3F/PO2R was highly sensitive (1 fg) when used in SYBR Green qPCR assays and is recommended for screening of plant materials potentially infected by P. omnivora or samples with suboptimal DNA quality. The described PCR-based detection methods will be useful for rapid and sensitive screening of infected plants in diagnostic laboratories, plant health inspections, and plant breeding programs.

First report of the association of a ‘Candidatus Phytoplasma ulmi’ isolate with a witches' broom disease of reed in China

Phragmites australis (Poaceae) is widely distributed around the world. It is not only a perennial weed, but also a raw material for the paper industry, a medicinal plant, animal fodder, an ornamental in landscaping and a building material. It now covers approximately two million ha in China, including ten thousand ha in Xinjiang Province. During September 2012, symptoms of witches' broom, stunting, and smaller leaves (Fig. 1A) were observed in over 57% of P. australis plants at the Shihezi University Experimental Station (Shihezi, Xinjiang Province). Leaf samples were collected from both symptomless plants and plants showing symptoms. Total DNA was extracted from 0.5 g fresh leaf midribs from P. australis plants with and without symptoms, using the CTAB method (Angelini et al., 1). The DNA extracts were tested for phytoplasma infection by a nested polymerase chain reaction (PCR) assay with phytoplasma universal 16S rDNA primer pairs P1/P7 (Deng & Hiruki, 2) and R16F2n/R16R2 (Gundersen & Lee, 3). The two primer pairs produced PCR products of 1.8 and 1.2 kb, respectively, from most of the plants with symptoms (4/5), but not from the symptomless plants (2/2). R16F2n/R16R2 amplicons were cloned (pMD18-T simple vector, TaKaRa, China) and sequenced. All of the phytoplasma R16F2n/R16R2 sequences were 100% identical to each other. BLAST comparisons showed that the P. australis witches' broom phytoplasma 16S rDNA consensus sequence (GenBank Accession No. KC331052) had 99% sequence identity with those of members of group 16SrV (‘Candidatus Phytoplasma ulmi’). Similarity coefficients and in silico restriction fragment length polymorphism (RFLP) analyses of the R16F2n/R16R2 sequence with AluI, BamHI, BfaI, BstUI, DraI, EcoRI, HaeIII, HhaI, HinfI, HpaI, HpaII, KpnI, Sau3AI, MseI, RsaI, I, and TaqI restriction endonucleases (iPhyClassifier, http://plantpathology.ba.ars.usda.gov/cgi-bin/resource/iphyclassifier.cgi) (Fig. 2), indicated that P. australis contained a phytoplasma strain identical to those of the reference strain AB052876, ‘Ca. Phytoplasma ulmi’, subgroup 16SrV-B. A neighbour-joining dendrogram (MEGA 5.0, USA) based on the 16S rDNA sequences of the P. australis witches' broom phytoplasma and twenty-five reference phytoplasmas in NCBI database supported the PCR and RFLP results since the P. australis witches' broom phytoplasma grouped within the 16SrV-B phytoplasma clade (Fig. 3). The group 16SrV was previously associated with witches' broom diseases of several plant species in China including jujube (Zhu et al., 6), paper mulberry (Liu et al., 4), and Chinese honeylocust (Min et al., 5). This is the first report of a ‘Ca. Phytoplasma ulmi‘-related strain affecting P. australis in China. This research was supported by the National Key Technology R&D Program of China (2011BAD48B00).

Risk analysis and development of a rapid method for identifying four species of Byssochlamys

Species of the genus Byssochlamys, heat-resistant fungi, cause the spoilage and poisoning of heat-processed acidic foods due to the formation of heat-resistant ascospores and the biosynthesis of patulin. Here we evaluated the heat resistance and patulin production of four species of Byssochlamys. Byssochlamys fulva was significantly more heat-resistant than the other species, and Byssochlamys lagunculariae and Byssochlamys nivea synthesized patulin. This is the first report of patulin production by B. lagunculariae. These findings indicate that the four species pose different health risks in food. Thus, we concluded that a method to identify the species of Byssochlamys is necessary. Identification at the species level based on morphology alone is difficult. We developed species-specific primers to identify B. nivea, B. fulva, B. lagunculariae, and Byssochlamys zollerniae using polymerase chain reaction (PCR) to amplify the β-tubulin gene. PCR using the primer sets designed for B. nivea (B. nivea1F/1R) amplified PCR products specific for B. nivea. Similarly, PCR using the B.fulva1F/1R, B.lag1F/1R and B.zol3F/R primer sets produced PCR products specific for B. fulva, B. lagunculariae, and B. zollerniae, respectively. PCR using these four primer sets did not detect other fungi involved in food spoilage and environmental contamination. This identification method is rapid and simple with extremely high specificity.

High Density Labeling of Polymerase Chain Reaction Products with the Fluorescent Base Analogue tCo

Fluorescent DNA of high molecular weight is an important tool for studying the physical properties of DNA and DNA-protein interactions, and it plays a key role in modern biotechnology for DNA sequencing and detection. While several DNA polymerases can incorporate large numbers of dye-linked nucleotides into primed DNA templates, the amplification of the resulting densely labeled DNA strands by polymerase chain reaction (PCR) is problematic. Here, we report a method for high density labeling of DNA in PCR reactions employing the 5'-triphosphate of 1,3-diaza-2-oxo-phenoxazine (tCo) and Deep Vent DNA polymerase. tCo is a fluorescent cytosine analogue that absorbs and emits light at 365 and 460 nm, respectively. We obtained PCR products that were fluorescent enough to directly visualize them in a gel by excitation with long UV light, thus eliminating the need for staining with ethidium bromide. Reactions with Taq polymerase failed to produce PCR products in the presence of only small amounts of dtCoTP. A comparative kinetic study of Taq and Deep Vent polymerase revealed that Taq polymerase, although it inserts dtCoTP with high efficiency opposite G, is prone to forming mutagenic tCo-A base pairs and does not efficiently extend base pairs containing tCo. These kinetics features explain the poor outcome of the PCR reactions with Taq polymerase. Since tCo substitutes structurally for cytosine, the presented labeling method is believed to be less invasive than labeling with dye-linked nucleotides and, therefore, produces DNA that is ideally suited for biophysical studies.

Rapid amplification for the detection of Mycobacterium tuberculosis using a non-contact heating method in a silicon microreactor based thermal cycler

This is the first report of a polymerase chain reaction (PCR)-based Mycobacterium tuberculosis diagnostic tool fabricated as a silicon microreactor for DNA amplification using a halogen lamp as the heating source. Our experiments demonstrate that PCR amplification can be successfully performed in silicon microreactors using a non-contact heating method. These devices have been used to amplify a specific fragment of the rpoB gene from M. tuberculosis, where mutations leading to multi-drug resistant strains are most likely to occur. Amplification was achieved in the microreactors in 34 min, compared to the 62 min amplification time required in a conventional peltier-heated PCR machine. Temperature control accuracy for our heating approach was ±1 °C. The amplification was performed from genomic DNA and produced PCR products in comparable quantities to a conventional thermal cycler, but in reduced time. This paper describes the fabrication, characterisation, control and use of these prototype devices for DNA amplification. PCR conditions were established using Lambda bacteriophage DNA as a model reaction system. Subsequently, DNA from the M. tuberculosis rpoB gene was successfully amplified.

Polymerase chain reaction with a primer pair in the 16S-23S rRNA spacer region for detection of Mycoplasma pulmonis in clinical isolates

To develop a diagnostic tool to identify Mycoplasma pulmonis ( M. pulmonis) in clinical isolates, we developed a polymerase chain reaction (PCR) assay using primers specific for the 16S-23S rRNA intergenic spacer region (SR) of M. pulmonis. One pair of PCR primers reacted specifically with two reference strains of M. pulmonis tested and seven samples isolated from naturally infected rats. The primer pair did not produce PCR products of the correct size from any other rodent or human mycoplasmas or cellular DNA from rodent lungs. Specificity of the PCR assay was confirmed by Southern blotting with probe specific for the SR of M. pulmonis. The PCR assay for detection of M. pulmonis established in this study is suitable for diagnosis of M. pulmonis infection in clinical cases.

Molecular detection of Fusarium solani f. sp. glycines in soybean roots and soil

A polymerase chain reaction (PCR)‐based method was developed to detect DNA of Fusarium solani f. sp. glycines, the cause of soybean sudden death syndrome. Two pairs of primers, Fsg1/Fsg2 designed from the mitochondrial small subunit ribosomal RNA gene, and FsgEF1/FsgEF2 designed from the translation elongation factor 1‐α gene, produced PCR products of 438 and 237 bp, respectively. Primer specificity was tested with DNA from 82 F. solani f. sp. glycines, 55 F. solani non‐SDS isolates, 43 isolates of 17 soybean fungal pathogens and the oomycete Phytophthora sojae, and soybean. The sensitivity of primer Fsg1/Fsg2 was 10 pg while that of FsgEF1/FsgEF2 was 1 ng when using F. solani f. sp. glycines total genomic DNA or down to 103 macroconidia g−1 soil. Nested PCR increased the sensitivity of the PCR assay 1000‐fold to 10 fg using primers Fsg1/Fsg2, and 1 pg using primers FsgEF1/FsgEF2. F. solani f. sp. glycines DNA was detected in field‐grown soybean roots and soil by PCR using either single pairs of primers or the combination of two pairs of primers. The occurrence of F. solani f. sp. glycines was determined using nested PCR for 47 soil samples collected from soybean fields in 20 counties of Illinois in 1999. F. solani f. sp. glycines was detected in soil samples from all five Illinois Agricultural Statistic Districts including 100, 89, 50, 92 and 50% of the samples from East, Central, North‐east and West Districts, respectively.

Molecular genetic analysis of archival gliomas using diagnostic smears.

Investigation of the clinical significance of genetic alterations in gliomas requires molecular genetic analysis using samples from retrospective or prospective clinical studies. However, diagnostic tissue is often severely limited and because of fixation, paraffin-embedded tissues (PET) contain degraded DNA. Intra-operative cytological preparations (smears) archived after diagnosis may represent an additional source of clinical material for genetic analysis. In this study, tissue samples were obtained by precision microdissection of archived diagnostic smears from 20 cases (1961-1999). All samples produced polymerase chain reaction (PCR) products for the beta globin gene, but the most recent samples amplified best and gave longer amplimers. For six cases, direct comparison was made between samples microdissected from smears and the corresponding PET. Samples from smears showed improved PCR performance and similar alleles on microsatellite marker analysis. One case, with smears of uninvolved cortex and tumour tissue available for microdissection, showed allelic imbalance at 10q23 on the basis of the smear results alone. PCR products from smears were shown to be suitable for direct sequence analysis (p53 gene). A PTEN mutation, found previously in an anaplastic astrocytoma by analysis of PET, was detected in the corresponding diagnostic smear. The results of this study indicate that tissue samples microdissected from diagnostic intra-operative cytological preparations may be suitable for molecular genetic analysis of gliomas.

Polymerase Chain Reaction (PCR)-based Typing Analysis of Atypical Isolates of the Fish Pathogen Aeromonas salmonicida

Two hundred and five isolates of atypical Aeromonas salmonicida, recovered from a wide range of hosts and countries were characterized by polymerase chain reaction (PCR) targeting four genes. The chosen genes were those encoding the extracellular A-layer protein (AP), the serine protease (Sprot), the glycerophospholipid:cholestrol acetyltransferase protein (GCAT), and the 16S rRNA (16S rDNA). All the atypical A. salmonicida isolates could be assigned to 4 PCR groups. Group 1 comprised 45 strains which tested positive for PCR amplification, using the 16S rDNA, GCAT2, Sprot2, and AP primer-sets. Group 2 comprised 88 strains with produced PCR products using the 16S rDNA, GCAT2 and AP primer-sets. Group 3 comprised 21 strains which produced PCR products using 16S rDNA, GCAT2 and Sprot2 primer-sets, and group 4 comprised 51 strains which produced PCR products using the 16S rDNA and GCAT2 primer-sets only. A. salmonicida subsp. salmonicida isolates tested, belonged to group 1. The PCR primer-sets separated A. salmonicida from other reference strains of Aeromonas species and related bacteria with the exception of Aeromonas hydrophila. The results indicated that PCR typing is a useful framework for characterization of the increasing number of isolations of atypical A. salmonicida.

Detection by PCR and analysis of the distribution of a fibronectin-binding protein gene (fbn) among staphylococcal isolates

The fibronectin-binding proteins of Staphylococcus aureus are considered to be important virulence factors for colonisation and infection. The polymerase chain reaction (PCR) was used to detect part of a gene equivalent to the fbnA gene of S. aureus in 120 isolates of staphylococci (S. aureus, S. epidermidis, S. haemolyticus, S. simulans, S. hominis, S. warneri, S. cohnii and S. lugdunensis). Primers specific for the binding domain region of the fbnA gene of S. aureus produced PCR products of the predicted sizes (93 and 207 bp). The identity of the PCR products was confirmed by digestion with DdeI and nucleic acid hybridisation. The fibronectin-binding activity of the staphylococci was determined with a particle agglutination assay (PAA). The fbn gene was found to be present by PCR in 107 of the 120 staphylococci tested, irrespective of their site of isolation, and expression of the gene was detected by PAA in 101 of the 120 strains.