Detection and quantitative analysis of zoospores of Pythium porphyrae, causative organism of red rot disease in Porphyra, by competitive PCR

The detection and quantitative analysis of Pythium porphyraezoospores was performed by PCR using PP-1 and PP-2 primers specific tothe internal transcribed spacer region of P. porphyrae. To estimatethe amount of fungal zoospores of P. porphyrae, an internal standardplasmid (pPPISC) containing a modified DNA fragment was constructed. Both ends of this fragment were complementary to the PCR primers. Amplification using primers PP-1 and PP-2 produced DNA fragments ofapproximately 700 and 400 bp from the target DNA of P. porphyraezoospores and from the pPPISC, respectively. To perform quantitativePCR, known quantities of pPPISC were added to reaction mixturescontaining the experimental DNAs extracted from zoospores. After aco-amplification reaction, the two different sized PCR products wereseparated by agarose gel electrophoresis and visualized by ethidium bromidestaining. The number of zoospores was estimated by comparing thefluorescence intensities of the PCR products using a charge-coupled deviceimage analyzer. The results show that competitive PCR using P.porphyrae specific primers and competitor pPPISC are useful tools for thequantitative analysis of P. porphyrae zoospores in seawater from Porphyra cultivation farms.     

Polymerase chain reaction/ligase detection reaction/hybridization assays using flow-through microfluidic devices for the detection of low-abundant DNA point mutations

We have microfabricated a flow-through biochip for the analysis of single base mutations in genomic DNA using two different materials: (1) a polycarbonate (PC) chip for performing a primary polymerase chain reaction (PCR) followed by an allele-specific ligation detection reaction (LDR) and (2) a poly(methyl methacrylate) (PMMA) chip for the detection of the LDR products using a universal array platform. The operation of the device was demonstrated by detecting low-abundant DNA mutations in gene fragments (K-ras) that carry point mutations with high diagnostic value for colorectal cancers. The PC microchip was used for sequential PCR/LDR in a continuous-flow format, in which the following three steps were carried out: (1) exponential amplification of gene fragments from genomic DNA; (2) mixing of the resultant PCR product with a LDR mixture via a Y-shaped passive micromixer and (3) ligation of two primers only when the particular mutation was present in the genomic DNA. A PMMA chip was employed as the microarray device, where zip code sequences (24-mer), which were complementary to sequences present on the discriminating primer, were micro-printed into fluidic channels embossed into the PMMA substrate. We successfully demonstrate the ability to detect one mutant DNA in 80 normal sequences with the integrated microfluidic device. The PCR/LDR/hybridization assay using the microchips performed the entire assay at a relatively fast processing speed: 18.7 min for PCR, 8.1 min for LDR, 5 min for hybridization, 10 min for washing and 2.6 min for fluorescence scanning (total processing time=ca. 50 min) with an order of magnitude reduction in reagents compared to bench-top formats.     

Molecular identification of Salmonella isolates from poultry and meat productsin Irbid City,Jordan

The sensitivity and accuracy of molecular diagnosis of Salmonella from meat and poultry products using polymerase chain reaction (PCR) was compared with conventional microbiological methods. A total of 212 samples representing the most frequently used fresh and frozen meat and poultry products (whole, cut, ground, and processed) were collected from different locations within the city of Irbid. DNA was extracted directly from each food sample and amplified using Salmonella-specific primers. Samples were also analysed using conventional microbiological methods for the presence of Salmonella spp. Results showed that Salmonella was detected in 185 samples out of 212 (87%) by PCR technique, while 172 (81%) samples were detected Salmonella positive by conventional microbiological methods. On the other hand, 27 (12.7%) samples were negative by PCR and 40 (18.8%) samples were negative by conventional microbiological methods. PCR assay proved to be an effective method for Salmonella detection in meat and poultry products with high specificity and sensitivity and more importantly a less time-consuming procedure. Using PCR, Salmonella spp. detection could be achieved within 24–36 h compared to 3–8 days for the conventional microbiological methods.     

Development of a PCR/Ligase Detection Reaction/Nanogold-Based Universal Array Approach for the Detection of Low-Abundant DNA Point Mutations

The aim of this study was to investigate the feasibility of combining PCR and ligase detection reaction (LDR) with a novel nano-gold-based universal array for the detection of low abundance point mutations from fetal DNA in maternal plasma samples. The sequence with the target point mutation was first amplified by PCR and then used as a template for LDR in which the upstream specific primer contains a tag sequence at the 5′-end. After hybridization to the probes of a universal array containing anti-tag sequences, the ligated products were bound to streptavidin-labeled nano-gold particles and the hybridization signals were amplified by silver staining. The PCR/LDR/universal array was first tested for sensitivity with nano-gold-based detection, and then this system was applied to detect the low abundance specific mutation IVS2 654(C→T) of the β-globin gene in a model using maternal plasma samples. The nano-gold-based method unambiguously identified a single mutation at a sensitivity of 1:1000. This approach was applied to detect the paternally inherited IVS2 654(C→T) mutation from thirty maternal plasma samples. The results were consistent with those obtained by PCR/reverse dot blot of amniotic fluid cell DNA. The PCR/LDR/nano-gold-based universal array is able to detect low-abundance point mutations with high sensitivity.     

A multiple PCR‐LDR assay for identification of two invasive cryptic species of whiteflies Bemisia tabaci

The MEAM1 and MED species of the cryptic species complex Bemisia tabaci are important invasive pests that cause tremendous crop losses worldwide. A rapid and highly reliable molecular technique is necessary to identify these species because they are morphologically indistinguishable. Therefore, a multiple polymerase chain reaction coupled with a ligase detection reaction (PCR‐LDR) that was based on polymorphisms in the mitochondrial cytochrome oxidase I (mtCOI) gene of B. tabaci was developed to distinguish the two cryptic species. An assessment of the method indicated that PCR‐LDR provided high specificity and sensitivity in discriminating MEAM1 (SHB) and MED (SHQ) whiteflies. In field tests, PCR‐LDR genotyping was performed in one 96‐well plate to identify 93 individuals collected from 8 districts in the suburbs of Shanghai. Complete concordance was observed between PCR‐LDR and sequencing methods. The method was used to confirm that MEAM1 and MED were found in two districts, but only the MED was found in the other six districts. PCR‐LDR, which is a transplantable platform, provides an alternative method for species identification of B. tabaci at low cost.     

Development of multisample detection system using a tag insertion primer and an electrochemical DNA chip

We have developed a novel multisample detection system by employing a technology combining a tag insertion primer and an electrochemical DNA chip. In the first application, Helicobacter species-infected mouse samples were detected. The primers that insert a different tag sequence in each sample were prepared, and loop-mediated isothermal amplification (LAMP) reaction was carried out. Then amplification products in which a part of the sequence was different in each sample could be obtained. The target sample in which these amplification products were mixed was injected into a cassette that included the DNA chip with immobilized probes. After the cassette was set in the DNA detection system, Genelyzer, the processes of hybridization, washing, and detection were performed by the system automatically. The positive and negative concordance rates of the existing nested polymerase chain reaction (PCR) method and this method were 100% (40/40 samples) and 97.3% (117/120 samples), respectively. This is a simple high-throughput method. Moreover, the cost per sample can be drastically lowered. Therefore, it is expected to contribute to the diagnosis of infectious agents in humans and animals.     

Analysis of single‐copy,nuclear microsatellite markers from flies collected on sticky traps

Sticky traps can provide large numbers of spatially referenced samples for use in molecular ecological studies of insects. However, the adhesives used on these traps, and the methods used to clean adhesive off trapped individuals, could potentially interfere with downstream molecular analyses. Specimens captured on sticky traps have been successfully used to analyse mitochondrial or multiple‐copy ribosomal DNA markers, but not single‐copy nuclear markers. Furthermore, the effects of trap adhesive and cleaning protocol on the success of molecular analyses have not been explored. Here, we examine the effects of trap adhesive, sample cleaning method, and sample storage condition on DNA concentration and purity, and on the ability to amplify single‐copy, nuclear microsatellite loci, using specimens of the western cherry fruit fly, Rhagoletis indifferens (Diptera: Tephritidae) captured on sticky traps in an orchard. We could extract DNA of high purity, and amplify microsatellite loci in multi‐plex polymerase chain reaction (PCR), under all combinations of treatments. However, DNA yield, DNA purity and the yield of PCR products were affected by treatment, with complex interactions among trap adhesive, sample cleaning method, and storage condition. Samples that were cleaned with acetone and stored dry had the highest DNA concentration. With respect to PCR amplification, samples cleaned with Histo‐clear produced much less product than those cleaned with acetone or not cleaned at all, whereas samples that were stored dry produced more PCR product than samples stored in ethanol. Insects captured on sticky traps can thus provide genetic data appropriate for molecular ecological analyses under a wide range of treatment conditions. However, potential interactions among adhesives, cleaning protocols and storage conditions suggest that any novel combination for treatment of samples from sticky traps should be tested on a small scale prior to collecting large numbers of samples for genetic studies.     

Universal DNA microarray method for multiplex detection of low abundance point mutations.

Cancers arise from the accumulation of multiple mutations in genes regulating cellular growth and differentiation. Identification of such mutations in numerous genes represents a significant challenge in genetic analysis, particularly when the majority of DNA in a tumor sample is from wild-type stroma. To overcome these difficulties, we have developed a new type of DNA microchip that combines polymerase chain reaction/ligase detection reaction (PCR/LDR) with "zip-code" hybridization. Suitably designed allele-specific LDR primers become covalently ligated to adjacent fluorescently labeled primers if and only if a mutation is present. The allele-specific LDR primers contain on their 5'-ends "zip-code complements" that are used to direct LDR products to specific zip-code addresses attached covalently to a three-dimensional gel-matrix array. Since zip-codes have no homology to either the target sequence or to other sequences in the genome, false signals due to mismatch hybridizations are not detected. The zip-code sequences remain constant and their complements can be appended to any set of LDR primers, making our zip-code arrays universal. Using the K- ras gene as a model system, multiplex PCR/LDR followed by hybridization to prototype 3x3 zip-code arrays correctly identified all mutations in tumor and cell line DNA. Mutations present at less than one per cent of the wild-type DNA level could be distinguished. Universal arrays may be used to rapidly detect low abundance mutations in any gene of interest.     

DNA amplification in the field: move over PCR,here comes LAMP

It would not be an exaggeration to say that among molecular technologies, it is PCR (polymerase chain reaction) that underpins the discipline of molecular ecology as we know it today. With PCR, it has been possible to target the amplification of particular fragments of DNA, which can then be analysed in a multitude of ways. The capability of PCR to amplify DNA from a mere handful of copies further means that conservationists and ecologists are able to sample DNA unobtrusively and with minimal disturbance to the environment and the organisms of interest. However, a key disadvantage of PCR‐based methods has been the necessity for a generally non‐portable, laboratory setting to undertake the time‐consuming thermocycling protocols. LAMP (loop‐mediated isothermal amplification) offers a logistically simpler protocol: a relatively rapid DNA amplification reaction occurs at one temperature, and the products are visualized with a colour change within the reaction tubes. In the first field application of LAMP for an ecological study, Centeno‐Cuadros et al. ( 2016 ) demonstrates how LAMP can be used to determine the sex of three raptor species. By enabling DNA amplification in situ and in ‘real‐time’, LAMP promises to revolutionize how molecular ecology is practised in the field.     

Relative quantification of chrysanthemum yellows (16Sr I) phytoplasma in its plant and insect host using real-time polymerase chain reaction

A real-time polymerase chain reaction (PCR) method for the quantification of chrysanthemum yellows (CY) phytoplasma DNA in its plant (Chrysanthemum carinatum) and insect (Macrosteles quadripunctulatus) host is described. The quantity of CY DNA was measured in each run relative to the amount of host DNA in the sample. Primers and a TaqMan probe for the specific PCR amplification of phytoplasma DNA were designed on a cloned CY-specific ribosomal fragment. Primers and TaqMan probes were also designed on sequences of the internal transcribed spacer region of the insect’s ITS1 rDNA and of the plant’s 18S rDNA for amplification from C. carinatum and M. quadripunculatus, respectively. Absolute quantification of CY DNA was achieved by comparison with a dilution series of the plasmid containing a CY 16S rDNA target sequence. Absolute quantification of plant and insect DNAs was achieved by comparison with a dilution series of the corresponding DNAs. Quantification of CY DNA in relation to host DNA was finally expressed as genome units (GU) of phytoplasma DNA per nanogram of host (plant or insect) DNA. Relative quantification avoided influences due to different yields during the DNA extraction procedure. The quantity of CY DNA was about 10,000–20,000 GU/ng of plant DNA and about 30,000–50,000 GU/ng of insect DNA. The method described could be used to phytoplasma multiplication and movement in different plant and insect hosts.     

用差异显示反转录PCR银染技术研究植物基因表达的差异

通过调整差异显示反转录PCR(DDRT-PCR)中总RNA、锚定引物、随机引物、cDNA和dNTP等关键试剂的用量,优化了适用于银染检测的DDRT-PCR方法.PCR扩增产物经6%变性聚丙烯酰胺凝胶垂直电泳分离后,银染能检测到多而清晰的条带.泳道中的条带数最少为40个,最多达80个,平均为60个,条带大小分布在100～900 bp范围,灵敏度为5 pg/mm² .此方法操作简便快速,灵敏度高,重复性好.采用这个改良的方法,分析了拟南芥野生型和ast突变型基因表达的差异.从16 000个cDNA扩增产物条带中筛选出28个差异条带.二次PCR扩增后,进一步筛选出13个差异条带,其中7个是野生型特异表达的,6个是突变型特异表达的,为进一步认识ast突变表型的产生机制奠定了基础.     

Restriction fragment length polymorphism analysis of polymerase chain reaction products amplified from mapped loci of rice (Oryza sativa L.) genomic DNA

Summary Thirty mapped Indica rice genomic (RG) clones were partially sequenced from each end. From such sequence data, pairs of oligonucleotides were synthesized to act as primers for polymerase chain reaction (PCR) amplification of the corresponding loci in crude total DNA preparations. The PCR products from DNA of Indica varieties were of the sizes expected from the sizes of the corresponding RG clones. However, size polymorphisms were seen between PCR products from Indica and Japonica varieties, and among wildOryza species. Restriction fragment length polymorphism (RFLP) was observed between PCR products of Indica varieties simply by electrophoretic analysis of restricted products, without the need for Southern hybridization or radiolabelling. The RFLPs noted between varieties ARC6650 and Phalguna were inherited in recombinant inbred lines derived from a cross between them. The RFLPs were detectable in PCR products amplified from DNA extracted by a simple procedure from single seedlings or leaves, and revealed genetic heterogeneity in cultivated lines. An approach is described that is relevant to the acceleration of classical plant breeding through molecular techniques.     

Electrical DNA-chip-based identification of different species of the genus Kitasatospora

The identification of different Kitasatospora strains has been shown with a DNA-chip based on an electrical readout scheme. The 16S-23S rDNA internal transcribed spacer region of these Actinomycetes was used for identification. Two different capture probes per strain were immobilized on the chip. The capture probes were spotted on a DNA-chip with electrode structures for an electrical DNA detection. A biotinylated PCR product of the 16S-23S rDNA region was incubated on the chips and bound to its complementary capture sequences. Followed by a gold nanoparticle or enzyme labeling and a deposition of silver, the binding of the PCR product was detected by an increase of the measured conductivity on the chip. To show the applicability of this detection system, four strains of Kitasatospora were chosen for an identification using the DNA-chip with electrical detection. Each strain was clearly identified using the system. Concentrations of the polymerase chain reaction (PCR) products within the range of 1 ng/ml to 1 μg/ml were detected and identified. These tests are the first application of this novel electrical detection scheme for the identification and classification of microorganisms. The presented results show that the DNA-chip with electrical detection can be used for a robust and cost-efficient DNA analysis.     

Integrated method for single-cell DNA extraction, PCR amplification, and sequencing of ribosomal DNA from harmful dinoflagellates Cochlodinium polykrikoides and Alexandrium catenella

A simplified technique was developed for DNA sequence-based diagnosis of harmful dinoflagellate species. This protocol integrates procedures for DNA extraction and polymerase chain reaction (PCR) amplification into a single tube. DNA sequencing reactions were performed directly, using unpurified PCR products as the DNA template for subsequent sequencing reactions. PCR reactions using DNA extracted from single cells of Cocodinium polykrikoides and Alexandrium catenella successfully amplified the target ribosomal DNA regions. DNA sequencing of the unpurified PCR products showed that DNA sequences corresponded to the expected locus of ribosomal DNA regions of both A. catenella and C. polykrikoides (each zero genetic distance and 100% sequence similarity). Using the protocol described in this article, there was little DNA loss during the purification step, and the technique was found to be rapid and inexpensive. This protocol clearly resolves the taxonomic ambiguities of closely related algal species (such as Alexandrium and Cochlodinium), and it constitutes a significant breakthrough for the molecular analysis of nonculturable dinoflagellate species.     

Extracting DNA from museum bird eggs, and whole genome amplification of archive DNA

We present a comprehensive protocol for extracting DNA from egg membranes and other internal debris recovered from the interior of blown museum bird eggs. A variety of commercially available DNA extraction methods were found to be applicable. DNA sequencing of polymerase chain reaction (PCR) products for a 176‐bp fragment of mitochondrial DNA was successful for most egg samples (> 78%) even though the amount of DNA extracted (mean = 14.71 ± 4.55 ng/µL) was significantly less than that obtained for bird skin samples (mean = 67.88 ± 4.77 ng/µL). For PCR and sequencing of snipe (Gallinago) DNA, we provide eight new primers for the ‘DNA barcode’ region of COI mtDNA. In various combinations, the primers target a range of PCR products sized from 72 bp to the full ‘barcode’ of 751 bp. Not all possible combinations were tested with archive snipe DNA, but we found a significantly better success rate of PCR amplification for a shorter 176‐bp target compared with a larger 288‐bp fragment (67% vs. 39%). Finally, we explored the feasibility of whole genome amplification (WGA) for extending the use of archive DNA in PCR and sequencing applications. Of two WGA approaches, a PCR‐based method was found to be able to amplify whole genomic DNA from archive skins and eggs from museum bird collections. After WGA, significantly more archive egg samples produced visible PCR products on agarose (56.9% before WGA vs. 79.0% after WGA). However, overall sequencing success did not improve significantly (78.8% compared with 83.0%).     

Preliminary genomic characterization of microsporidian Nosema bombycis

In order to characterize the genome of Nosema bombycis, the techniques of karyotyping, pulsed field gel electrophoresis, and polymerase chain reaction were applied. Nosema genomic DNA moved as 23 kb fragment on a standard agarose gel. The karyotype showed four chromosomes, the molecular karyotyping by pulsed field gel electrophoresis also showed four chromosomes. Arbitrarily primed polymerase chain reaction (PCR) with various primers showed amplification products of sizes ranging from 1.6 to 0.15 kb. Polymerase chain reaction with specific primer showed an amplification product of approximately 315 nucleotides. The DNA hybridizations are discussed. This is the first report of its kind on microsporidian Nosema bombycis. The current data can play a major role in elucidating the molecular biology of this parasite. This revised version was published online in July 2006 with corrections to the Cover Date.     

Universal DNA array detection of small insertions and deletions in BRCA1 and BRCA2

Array-based mutation detection methodology typically relies on direct hybridization of the fluorescently labeled query sequence to surface-bound oligonucleotide probes. These probes contain either small sequence variations or perfect-match sequence. The intensity of fluorescence bound to each oligonucleotide probe is intended to reveal which sequence is perfectly complementary to the query sequence. However, these approaches have not always been successful, especially for detection of small frameshift mutations. Here we describe a multiplex assay to detect small insertions and deletions by using a modified PCR to evenly amplify each amplicon (PCR/PCR), followed by ligase detection reaction (LDR). Mutations were identified by screening reaction products with a universal DNA microarray, which uncouples mutation detection from array hybridization and provides for high sensitivity. Using the three BRCA1 and BRCA2 founder mutations in the Ashkenazi Jewish population (BRCA1 185delAG; BRCA1 5382insC; BRCA2 6174delT) as a model system, the assay readily detected these mutations in multiplexed reactions. Our results demonstrate that universal microarray analysis of PCR/PCR/LDR products permits rapid identification of small insertion and deletion mutations in the context of both clinical diagnosis and population studies.     

DNA probes for the Y-chromosome ofSilene latifolia,a dioecious angiosperm

Summary In order to obtain markers for the Y chromosome ofSilene latifolia, we pooled equal weights of leaf tissue from 18 female siblings into one sample and repeated the process with 18 male siblings. Pooling was intended to provide a common genetic background for each sample, leaving the absence or presence of the Y chromosome as the primary difference between the two samples. DNA was extracted from each sample and subjected to polymerase chain reaction (PCR) amplification with arbitrary 10 bp primers. Four of 60 primers used gave an amplification with the male DNA not found among those from the female DNA. Each of these was subsequently shown to provide a reliable marker for the Y chromosome.     

Rapid detection of viable bacteria by nested polymerase chain reaction via long DNA amplification after ethidium monoazide treatment

In assays to determine whether viable cells of Enterobacteriaceae are present in pasteurized milk, the typical ethidium monoazide (EMA) polymerase chain reaction (PCR) targets a short stretch of DNA. This process often triggers false-positive results owing to the high level of dead cells of Enterobacteriaceae that had initially contaminated the sample. We have developed a novel, direct, real-time PCR that does not require DNA isolation (DQ-PCR) to detect low levels of cells of Enterobacteriaceae regardless of live and dead cells first. We confirmed that the DQ-PCR targeting a long DNA (the 16S ribosomal RNA [rRNA] gene, amplified length of 1514 bp) following EMA treatment is a promising tool to detect live bacteria of all genera owing to the complete suppression of background signal from high levels of dead bacteria in pasteurized milk. However, when identifying viable bacteria in pasteurized milk, commercial PCR primers designed for detecting long stretches of DNA are generally not available. Thus, we treated samples with EMA and then carried out an initial round of PCR of a long stretch of DNA (16S gene, 1514 bp). We then performed another round of PCR, a novel nested PCR to generate short products using commercial primers. This procedure resulted in the rapid detection of low levels of viable cells of Enterobacteriaceae.     

Optimized conditions for rapd analysis inPinus radiata

Summary Pinus radiata is the most important softwood plantation species in Australia and New Zealand. The improtance of this species in forestry has led to an increasing demand to improve the efficiency of selection time of the production population, which currently takes 13 yr by traditional methods. With the application of molecular biology techniques such as random amplified polymorphic DNA (RAPD) the selection period can be reduced to 6 yr. In this study, the conditions for RAPD were optimized and the feasibility of this marker system was investigated with different families ofPinus radiata from Tasmania and South Australia. Best concentrations of Taq-polymerase (1 U), magnesium chloride (2 mM), and template DNA (20 ng) were selected to test different polymerase chain reaction (PCR) thermocycler profiles. Devey's et al. (1996) program was the most effective for production of clear RAPD bands. Best conditions were investigated to screen 10–12 bp arbitrary Breasatec and Operon primers. Both types were found useful at detecting genetic variation between families. Seventy percent and thirty percent of the selected Bresatec and Operon primers, respectively, produced polymorphic bands.