PCR Primer Pairs Research Articles

Occurrence and Partial Characterization of Lettuce big vein associated virus and Mirafiori lettuce big vein virus in Lettuce in Iran.

Mirafiori lettuce big vein virus (MiLBVV) and lettuce big vein associated virus (LBVaV) were found in association with big vein disease of lettuce in Iran. Analysis of part of the coat protein (CP) gene of Iranian isolates of LBVaV showed 97.1-100% nucleotide sequence identity with other LBVaV isolates. Iranian isolates of MiLBVV belonged to subgroup A and showed 88.6-98.8% nucleotide sequence identity with other isolates of this virus when amplified by PCR primer pair MiLV VP. The occurrence of both viruses in lettuce crop was associated with the presence of resting spores and zoosporangia of the fungus Olpidium brassicae in lettuce roots under field and greenhouse conditions. Two months after sowing lettuce seed in soil collected from a lettuce field with big vein affected plants, all seedlings were positive for LBVaV and MiLBVV, indicating soil transmission of both viruses.

Development and application of specific polymerase chain reaction assay targeting the gyrB gene for rapid detection of Riemerella anatipestifer

A pair of PCR primers was designed and synthesized to amplify a gyrB gene sequence from Riemerella anatipestifer (RA). A fragment of 194 bp was detected in RA-positive isolates, whereas other isolates were negative, which confirmed the high specificity of the primers and PCR conditions. As little as 1.6 × 10(4) cfu/mL of cultural liquid was required by this method. We compared a 16S rRNA sequence-based PCR method and a Biolog bacterial identification system used in the detection and identification of suspicious isolates of RA in clinical tests. The results showed that the gyrB-based PCR was consistent with the results of the Biolog identification system and was more specific. By applying the gyrB-PCR to detect RA strains in 56 duck livers, a positive rate of 46% (26/56) was observed, whereas the positive rate of 85 throat swabs from clinically healthy ducks was 11%. Thus, this method could be used for the epidemiological investigation and preliminary isolate identification of RA.

Authentication of ‘Cereza del Jerte’ cherry cultivars using real time PCR

The purpose of this work was to develop a real-time polymerase chain reaction method for the determination of sweet cherry cultivars for the authentication of “Cereza del Jerte”. Three autochthonous cultivars of the “Picota” type sweet cherry, ‘Ambrunés’, ‘Pico Negro’, and ‘Pico Colorado’, and the foreign cultivar ‘Sweetheart’ were used in the study. Three molecular markers were obtained through analysis of RAPD profiles with OPD11 primer, after testing 27 primers, one of 822 bp for the ‘Pico Negro’ cultivar and two markers of 763 bp and 635 bp for the ‘Ambrunés’ cultivar. Those markers were then used to design new primer pairs for real-time PCR with SYBR® Green, one of which allowed the Picota type cherry cultivars to be distinguished from the Sweetheart cultivar since the amplification products had distinct melting temperatures.

Analyzing the gene expression profile of pediatric acute myeloid leukemia with real-time PCR arrays

BackgroundThe Real-time PCR Array System is the ideal tool for analyzing the expression of a focused panel of genes. In this study, we will analyze the gene expression profile of pediatric acute myeloid leukemia with real-time PCR arrays.MethodsReal-time PCR array was designed and tested firstly. Then gene expression profile of 11 pediatric AML and 10 normal controls was analyzed with real-time PCR arrays. We analyzed the expression data with MEV (Multi Experiment View) cluster software. Datasets representing genes with altered expression profile derived from cluster analyses were imported into the Ingenuity Pathway Analysis Tool.ResultsWe designed and tested 88 real-time PCR primer pairs for a quantitative gene expression analysis of key genes involved in pediatric AML. The gene expression profile of pediatric AML is significantly different from normal control; there are 19 genes up-regulated and 25 genes down-regulated in pediatric AML. To investigate possible biological interactions of differently regulated genes, datasets representing genes with altered expression profile were imported into the Ingenuity Pathway Analysis Tool. The results revealed 12 significant networks. Of these networks, Cellular Development, Cellular Growth and Proliferation, Tumor Morphology was the highest rated network with 36 focus molecules and the significance score of 41. The IPA analysis also groups the differentially expressed genes into biological mechanisms that are related to hematological disease, cell death, cell growth and hematological system development. In the top canonical pathways, p53 and Huntington’s disease signaling came out to be the top two most significant pathways with a p value of 1.5E-8 and2.95E-7, respectively.ConclusionsThe present study demonstrates the gene expression profile of pediatric AML is significantly different from normal control; there are 19 genes up-regulated and 25 genes down-regulated in pediatric AML. We found some genes dyes-regulated in pediatric AML for the first time as FASLG, HDAC4, HDAC7 and some HOX family genes. IPA analysis showed the top important pathways for pediatric AML are p53 and Huntington’s disease signaling. This work may provide new clues of molecular mechanism in pediatric AML.

Development of EST-SSR PCR primers within three North Pacific rockfishes of the genus Sebastes

Severe declines in abundance of Pacific Ocean Perch (Sebastes alutus) and other rockfishes have led to catch restrictions along the US west coast. The stock structure of many important commercial members of Sebastes spp remains a major source of uncertainty in stock assessment models. Here we report development of PCR primer pairs from 13 novel EST-SSR loci obtained from public Sebastes EST databases, and redesigned primer pairs from six published EST-SSR loci. Amplification and diversity data from these and 27 published EST-SSR loci are reported for S. polyspinis, S. alutus, and two cryptic sub-types of S. aleutianus. Loci were moderately polymorphic, with heterozygosities averaging 0.44, and allele number averaging 5.2 across all loci and species. Divergence between the two S. aleutianus sub-types was high (mean F ST = 0.33) allowing for unambiguous identification. The loci will be informative for resolving stock structure issues and possible local adaptation along the species’ ranges.

Predicting family-wide consensus sequence of pre-mRNA splicing signals in legumes and their potential utility as phylogenetic markers

Recent studies have shown that an intron is not merely “junk”, but something that plays important roles in many biological processes such as gene expression regulation and alternative splicing. For purposes of studying intron structures and predicting consensus splice motifs, a total of 102 legume species were used to isolate introns across the family. Of 196 gene-targeted PCR primer pairs, we successfully amplified 118 intron-containing genes (60.2 %) and obtained a total of 1,870 introns with an average size of 143 nucleotides, ranging from 61 to 1,036. Species-based compilation of 5′- and 3′-splicing motifs showed, to some extent, lineage-specific conservation in each splicing motif. Compilation of the entire intron set permitted prediction of the consensus sequences of splicing signal motifs in legumes, AYGWGTABABGH and TVNC/TAGGHTV for the 5′SS and 3′SS, respectively. Interestingly, these consensus motifs are very similar to the corresponding genome-wide splicing signals of two model systems, Arabidopsis and rice. This result suggests conservation of pre-mRNA splicing mechanism occurring in higher plants. Multiple alignments of CALTL introns demonstrated that the BP-3′SS region was relatively more conserved than the 5′SS-BP region. We speculate that length of the BP-3′SS region needs to be retained for the interaction with U2AF protein. Phylogenetic analysis demonstrates that each of three splicing motifs is not only phylogenetically informative, but also relevant to evolutionary divergence of species. This result suggests that the splice signal sequences would be a useful tool for the molecular phylogenetic analysis. We also anticipate that gene-targeted amplification in multiple genomes, described in this study, would facilitate studies on intron-located functional elements involved in gene expression regulations.

Quantitative RT-PCR profiling of the Rabbit Immune Response: Assessment of Acute Shigella flexneri Infection

Quantitative reverse transcription PCR analysis is an important tool to monitor changes in gene expression in animal models. The rabbit is a widely accepted and commonly used animal model in the study of human diseases and infections by viral, fungal, bacterial and protozoan pathogens. Only a limited number of rabbit genes have, however, been analyzed by this method as the rabbit genome sequence remains unfinished. Recently, increasing coverage of the genome has permitted the prediction of a growing number of genes that are relevant in the context of the immune response. We hereby report the design of twenty-four quantitative PCR primer pairs covering common cytokines, chemoattractants, antimicrobials and enzymes for a rapid, sensitive and quantitative analysis of the rabbit immune response. Importantly, all primer pairs were designed to be used under identical experimental conditions, thereby enabling the simultaneous analysis of all genes in a high-throughput format. This tool was used to analyze the rabbit innate immune response to infection with the human gastrointestinal pathogen Shigella flexneri. Beyond the known inflammatory mediators, we identified IL-22, IL-17A and IL-17F as highly upregulated cytokines and as first responders to infection during the innate phase of the host immune response. This set of qPCR primers also provides a convenient tool for monitoring the rabbit immune response during infection with other pathogens and other inflammatory conditions.

Aspergillus section Versicolores: nine new species and multilocus DNA sequence based phylogeny

β-tubulin, calmodulin, internal transcribed spacer and partial lsu-rDNA, RNA polymerase 2, DNA replication licensing factor Mcm7, and pre-rRNA processing protein Tsr1 were amplified and sequenced from numerous isolates belonging to Aspergillus sect. versicolor. The isolates were analyzed phylogenetically using the concordance model to establish species boundaries. Aspergillus austroafricanus, A. creber, A. cvjetkovicii, A. fructus, A. jensenii, A. puulaauensis, A. subversicolor, A. tennesseensis and A. venenatus are described as new species and A. amoenus, A. protuberus,A. sydowii, A. tabacinus and A. versicolor are accepted as distinct species on the basis of molecular and phenotypic differences. PCR primer pairs used to detect A. versicolor in sick building syndrome studies have a positive reaction for all of the newly described species except A. subversicolor.

Development of Exon-Primed Intron-Crossing (EPIC) PCR primers for the malaria vector Anopheles pseudopunctipennis (Diptera: Culicidae)

Using the Anopheles gambiae Giles genome as a template, we designed, screened and identified 14 novel Exon-Primed Intron-Crossing (EPIC) PCR primer pairs for Anopheles pseudopunctipennis Theobald 1901, a major vector of human Plasmodium sp. in South America. These primers were designed to target the conserved regions flanking consecutive exons of different genes and enabled the amplification of 17 loci of which nine were polymorphic. Polymorphisms at these loci ranged from two to four alleles. Intron length polymorphism analysis is a useful tool, which will allow the study of the population structure of this mosquito species, which remains poorly understood.

Detection of heartworm infection in dogs via PCR amplification and electrospray ionization mass spectrometry of nucleic acid extracts from whole blood samples

To develop and evaluate a rapid and accurate assay involving PCR amplification and electrospray ionization mass spectrometry of nucleic acid extracts from whole blood samples for the detection of Dirofilaria immitis infection in dogs. Whole blood nucleic acid extracts from 29 dogs experimentally infected with D immitis (and in which circulating D immitis antigen was detected) and 10 uninfected dogs. 16 of the 29 whole blood samples from infected dogs were examined at the time of collection for circulating microfilaria. Nucleic acids were extracted from all whole blood specimens and underwent PCR amplification with 12 PCR primer pairs designed to detect a wide range of pathogens (including the Wolbachia endosymbiont of D immitis) and electrospray ionization mass spectrometry. On the basis of assay results, heartworm infection was detected in 13 of 13 antigen-positive dogs of unknown microfilaria status, 11 of 11 antigen-positive dogs with circulating microfilaria, 0 of 3 antigen-positive dogs tested at 3 months after larval infection, 0 of 2 antigen-positive dogs with occult infections, and 0 of 10 uninfected dogs. With the assay under investigation, it was possible to identify D immitis infection in dogs with circulating microfilaria via detection of the obligate Wolbachia endosymbiont of D immitis. It was not possible to identify dogs with occult infections, which suggested that circulating microfilaria must be present to detect infection with this assay, although further studies would be required to verify that finding.

Detection of <i>Cryptosporidium parvum</i> and <i>Giardia lamblia</i> in Water Supply by Quantitative Real-Time PCR

Cryptosporidium parvum and Giardia lamblia are common pathogenic protozoa in water, which pose high risk to drinking water supply. In the present study, detection of C. parvum and G. lamblia was performed by quantitative real-time PCR (RT-PCR). Pairs of PCR primers were evaluated for the detection specificity to pathogenic C. parvum and G. lamblia. The recovery of the RT-PCR detection procedure was examined and high recovery rates (i.e., more than 45％ for C. parvum and more than 50％ for G. lamblia ) were achieved. The RT-PCR method was used to detect C. parvum and G. lamblia in a secondary water supply. The results indicated the potential application of the quantitative RT- PCR method in detection of C. parvum and G. lamblia in water supply.

Sequetyping: Serotyping Streptococcus pneumoniae by a Single PCR Sequencing Strategy

The introduction of pneumococcal conjugate vaccines necessitates continued monitoring of circulating strains to assess vaccine efficacy and replacement serotypes. Conventional serological methods are costly, labor-intensive, and prone to misidentification, while current DNA-based methods have limited serotype coverage requiring multiple PCR primers. In this study, a computer algorithm was developed to interrogate the capsulation locus (cps) of vaccine serotypes to locate primer pairs in conserved regions that border variable regions and could differentiate between serotypes. In silico analysis of cps from 92 serotypes indicated that a primer pair spanning the regulatory gene cpsB could putatively amplify 84 serotypes and differentiate 46. This primer set was specific to Streptococcus pneumoniae, with no amplification observed for other species, including S. mitis, S. oralis, and S. pseudopneumoniae. One hundred thirty-eight pneumococcal strains covering 48 serotypes were tested. Of 23 vaccine serotypes included in the study, most (19/22, 86%) were identified correctly at least to the serogroup level, including all of the 13-valent conjugate vaccine and other replacement serotypes. Reproducibility was demonstrated by the correct sequetyping of different strains of a serotype. This novel sequence-based method employing a single PCR primer pair is cost-effective and simple. Furthermore, it has the potential to identify new serotypes that may evolve in the future.

Abstract LB-410: Targeted resequencing of genes frequently mutated in esophageal adenocarcinoma using highly multiplexed Ion Torrent AmpliSeqTM technology and semiconductor sequencing

Abstract Introduction: Whole genome and whole exome sequencing by large collaborative research groups is providing us with an unprecedented view of the mutations that occur in cancer. Identifying clinically-relevant and actionable mutations however remains a challenge that will most likely be met by individual groups focused on specific clinical questions and scenarios. Such studies will rely on targeted resequencing of selected genes in large numbers of well-annotated clinical specimens. These specimens are most often formalin-fixed and paraffin-embedded (FFPE) and some of the most valuable samples are small biopsies or fine needle aspirates (FNA's). Translational and clinical researchers require rapid and flexible resequencing approaches that are compatible with the small amounts of highly degraded nucleic acids derived from these challenging samples. Materials & Methods: Using Illumina whole exome sequence data from >60 esophageal adenocarcinoma (EAC) specimens, we identified a set of 20 genes that are frequently mutated in this tumor. PCR amplicons no larger than 140bp were designed to cover all exons from these genes, using the Ion Torrent AmpliSeqTM Designer pipeline. Primers were pooled for a 1285-plex multiplex PCR preamplification step starting with 1-20ng of input DNA from both FFPE and frozen tissue and the resulting amplicon products were used to generate sequencing libraries. Semiconductor sequencing was performed on the Ion Personal Genome MachineTM (PGMTM) sequencer. Metrics evaluated included reads on target, base accuracy, uniform coverage of bases and sequencing base read depth. Mutations detected in EAC samples were compared between the two sequencing platforms. Data from dysplastic and metaplastic esophageal biopsies are now being generated to identify mutations that occur in the development of EAC. Results: 1285 PCR primer pairs were designed that covered 66103bp of the coding region of the 20 target genes. Sequencing data covered >99% with at least 100 reads indicating minimal drop-out during preamplification. Average base read depth was >1900X and no significant differences were observed between FFPE and frozen tissue derived DNA. Initial studies demonstrate 100% concordance of mutations (n=42) detected in the 20 target genes and mutant allele frequencies are highly correlated between the two sequencing approaches (slope =0.96; R2 =0.85; range 4-78%). Conclusion: We have used the Ion Torrent AmpliSeqTM technology to detect mutations in EAC specimens and precursor lesions with high sensitivity and accuracy. The flexible primer design pipeline combined with AmpliSeqTM technology provides a fast and easy approach to targeted resequencing in particularly challenging clinical specimens including FFPE biopsies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-410. doi:1538-7445.AM2012-LB-410

Effects Of Orange Juice And Vitamin C On The Expression Of Some Selected Genes ( Tnfr, Sod3, P53, Pgr ) In Sprague‐Dawley Rat Brain

The comparative effects of Vitamin C and Nigerian local orange juice on the gene expression levels of Superoxide dismutase (Sod), Tumour necrosis Factor Receptor (Tnfr), p53 protein, and progesterone receptor (Pgr) genes were evaluated in rat neuronal tissues.5ml/kg bodyweight of extracted orange juice and 20mg/kg bodyweight Vit C were administered orally to Sprague Dawley rats, which were divided into control, Vit. C and orange groups. After the 7th day of administration, total RNA was extracted from the rat brains and normalized RNA was converted to cDNA. PCR Primer pairs were designed using Primer 3 and NCBI primer blast tool. Sybr green Real time PCR relative quantification was done.Orange juice extract contains 15.84 mg Vit C, 13.67g total sugar, 0.02 mg Sodium, 0.15 mg Iron, 11.48 mg Magnesium, 44.20 mg Calcium, 4.76 mg Potassium, 0.75 mg Zinc, and 22.9 mg Phosphorus. Relative quantification gene expression assays in the rats show a 400 fold change in Tnfr and 38 fold change in p53 gene in the orange administered group in comparison to the Vit C administered and control groups. Vit C caused a 420 fold change in the expression of Pgr gene in comparison with the orange administered group. Data show that orange juice administered at 5ml/kg body weight show higher levels of gene expressions of Tnfr and p53 genes, thereby indicating brain tumour preventing ability of oranges.

Development of intron length polymorphism markers in cowpea [Vigna unguiculata (L.) Walp.] and their transferability to other Vigna species

Expressed sequence tag (EST) sequences available in the public databases provide a cost-effective and valuable genomic resource for the development of molecular markers. Introns which are non-coding DNA sequences of the gene could be used as potential molecular markers as they are highly variable compared to the coding sequences. This study reports the development of intron length polymorphism markers in cowpea [Vigna unguiculata (L.) Walp.]. The ESTs of cowpea were aligned with genomic sequences of Arabidopsis and soybean to predict the position and number of introns in cowpea. Of the 110 PCR primer pairs designed to amplify the intronic regions, 98 primer pairs resulted in successful amplification and were identified as cowpea intron length polymorphism (CILP) markers. Out of the 45 randomly selected CILP markers, 36 % markers produced length variation in the ten cowpea genotypes, collectively yielding 33 alleles with an average of 2.0 alleles/locus. The polymorphism information content of the CILP markers ranged from 0.18 to 0.64 with an average of 0.34. Of the 98 CILP markers, 93 markers (95 %) showed transferability to other Vigna species. Dendrograms based on CILP markers clearly distinguished the cowpea genotypes as well as other Vigna species, demonstrating the utility of CILP markers in genetic diversity and phylogenetic studies. These CILP markers will be very useful in the genome analysis and marker-assisted breeding of cowpea and other Vigna species.

Tagging and mapping candidate loci for vernalization and flower initiation in hexaploid oat

Flowering time is a decisive factor in the adaptation of oat. Some oat varieties require low temperatures for floral initiation, a process called vernalization. The objectives of this study were to clone, characterize, and map genes associated with vernalization in oat, and to identify markers linked to quantitative trait loci (QTL) that affect vernalization response. Genetic linkage maps were developed using Diversity Arrays Technology markers in recombinant inbred lines from the oat populations UFRGS 8 × UFRGS 930605 and UFRGS 881971 × Pc68/5*Starter. Flowering time and response to vernalization were characterized using field trials and controlled greenhouse experiments, and QTL were identified in two genetic regions on each of the two maps. PCR primer pairs anchored in the conserved coding regions of the Vrn1, Vrn2, and Vrn3 genes from wheat, barley, and Lolium were used to amplify and clone corresponding oat sequences. Cloned sequences corresponding to the targeted genes were recovered for both Vrn1 and Vrn3. A copy of the Vrn3 gene was mapped using a PCR amplicon, and an oat Vrn1 fragment was mapped by restriction fragment length polymorphism analysis. The location of the mapped Vrn1 locus was homologous to major QTL affecting flowering time in other work, and homoeologous to major QTL affecting response to vernalization in this study.

Diversity of flavin-binding monooxygenase genes (almA) in marine bacteria capable of degradation long-chain alkanes

Many bacteria have been reported as degraders of long-chain (LC) n-alkanes, but the mechanism is poorly understood. Flavin-binding monooxygenase (AlmA) was recently found to be involved in LC-alkane degradation in bacteria of the Acinetobacter and Alcanivorax genera. However, the diversity of this gene and the role it plays in other bacteria remains unclear. In this study, we surveyed the diversity of almA in marine bacteria and in bacteria found in oil-enrichment communities. To identify the presence of this gene, a pair of degenerate PCR primers were was designed based on conserved motifs of the almA gene sequences in public databases. Using this approach, we identified diverse almA genes in the hydrocarbon-degrading bacteria and in bacterial communities from the surface seawater of the Xiamen coastal area, the South China Sea, the Indian Ocean, and the Atlantic Ocean. As a result, almA was positively detected in 35 isolates belonging to four genera, and a total of 39 different almA sequences were obtained. Five isolates were confirmed to harbor two to three almA genes. From the Xiamen coastal area and the Atlantic Ocean oil-enrichment communities, a total of 60 different almA sequences were obtained. These sequences mainly formed two clusters in the phylogenetic tree, named Class I and Class II, and these shared 45-56% identity at the amino acid level. Class I contained 11 sequences from bacteria represented by the Salinisphaera and Parvibaculum genera. Class II was larger and more diverse, and it was composed of 88 sequences from Proteobacteria, Gram-negative bacteria, and the enriched bacterial communities. These communities were represented by the Alcanivorax and Marinobacter genera, which are the two most popular genera hosting the almA gene. AlmA was also detected across a wide geographical range, as determined by the origin of the bacterial host. Our results demonstrate the diversity of almA and confirm its high rate of occurrence in hydrocarbon-degrading bacteria, indicating that this gene plays an important role in the degradation of LC alkanes in marine environments.

PhiSiGns: an online tool to identify signature genes in phages and design PCR primers for examining phage diversity

BackgroundPhages (viruses that infect bacteria) have gained significant attention because of their abundance, diversity and important ecological roles. However, the lack of a universal gene shared by all phages presents a challenge for phage identification and characterization, especially in environmental samples where it is difficult to culture phage-host systems. Homologous conserved genes (or "signature genes") present in groups of closely-related phages can be used to explore phage diversity and define evolutionary relationships amongst these phages. Bioinformatic approaches are needed to identify candidate signature genes and design PCR primers to amplify those genes from environmental samples; however, there is currently no existing computational tool that biologists can use for this purpose.ResultsHere we present PhiSiGns, a web-based and standalone application that performs a pairwise comparison of each gene present in user-selected phage genomes, identifies signature genes, generates alignments of these genes, and designs potential PCR primer pairs. PhiSiGns is available at (http://www.phantome.org/phisigns/; http://phisigns.sourceforge.net/) with a link to the source code. Here we describe the specifications of PhiSiGns and demonstrate its application with a case study.ConclusionsPhiSiGns provides phage biologists with a user-friendly tool to identify signature genes and design PCR primers to amplify related genes from uncultured phages in environmental samples. This bioinformatics tool will facilitate the development of novel signature genes for use as molecular markers in studies of phage diversity, phylogeny, and evolution.

Evaluation of DNA Extraction Methods and Their Clinical Application for Direct Detection of Causative Bacteria in Continuous Ambulatory Peritoneal Dialysis Culture Fluids from Patients with Peritonitis by Using Broad-Range PCR

BackgroundThe aims of this study were to compare several DNA extraction methods and 16S rDNA primers and to evaluate the clinical utility of broad-range PCR in continuous ambulatory peritoneal dialysis (CAPD) culture fluids.MethodsSix type strains were used as model organisms in dilutions from 108 to 100 colony-forming units (CFU)/mL for the evaluation of 5 DNA extraction methods and 5 PCR primer pairs. Broad-range PCR was applied to 100 CAPD culture fluids, and the results were compared with conventional culture results.ResultsThere were some differences between the various DNA extraction methods and primer sets with regard to the detection limits. The InstaGene Matrix (Bio-Rad Laboratories, USA) and Exgene Clinic SV kits (GeneAll Biotechnology Co. Ltd, Korea) seem to have higher sensitivities than the others. The results of broad-range PCR were concordant with the results from culture in 97% of all cases (97/100). Two culture-positive cases that were broad-range PCR-negative were identified as Candida albicans, and 1 PCR-positive but culture-negative sample was identified as Bacillus circulans by sequencing. Two samples among 54 broad-range PCR-positive products could not be sequenced.ConclusionsThere were differences in the analytical sensitivity of various DNA extraction methods and primers for broad-range PCR. The broad-range PCR assay can be used to detect bacterial pathogens in CAPD culture fluid as a supplement to culture methods.

Molecular Cloning of Promoter of <i>AP3</i> Gene from <i>Arabidopsis thaliana</i> Ecotype (Col) and Construction of the Plant Expression Vector

Belonging to MADS-Box gene family, APETALA3 ( AP3 ) gene of Arabidopsis floral organ class B is expressed in petals and stamens specifically. AP3 gene which encodes a transcription factor controls the development of petals and stamens of dicotyledons by cooperation with class A and class C genes, respectively. The results showed that the promoter of AP3 was the flower-specific expression promoter. Therefore, the cloning and functional identification of AP3 gene promoter from Arabidopsis thaliana will play significant effects on the oriented improvement of the commercial traits related to the flowers of ornamental plants. Here, a pair of specific PCR primers was designed according to the promoter sequence of AP3 gene of Arabidopsis thaliana ecotype (Ler) reported in GenBank (U30729). A length of 1 767 bp of AP3 gene promoter was obtained from Arabidopsis thaliana ecotype (Col) by using PCR technique with high-fidelity DNA polymerase KOD-plus, named as pAtAP3 , the GenBank accession No. is FJ619533. Online Bl2seq analysis indicated that the sequence similarity between pAtAP3 and U30729 was 98%. It also showed that the sequence similarity between pAtAP3 and the BAC clone T12E18 (AL132971) of Arabidopsis thaliana ecotype (Col) from 9 264 to 11 030 was up to 100%, and its downstream sequence encoded AP3 protein (CAB81799), which approved that the sequence we cloned was the promoter of AP3 gene of Arabidopsis thaliana ecotype (Col). Online PLACE analysis displayed that pAtAP3 contained the basic cis -elements of promoter such as TATA-box and CAAT-box. It also comprised several cis -elements related to flower-specific expression, such as CArG1, CArG2, CArG3 and anther-box. Moreover, plant expression vector of pAtAP3 :: GUS was successfully constructed in this research, which would lay the foundation for functional identification of the AP3 promoter of Arabidopsis thaliana ecotype (Col).