Anonymous DNA Fragments Research Articles

Molecular detection of Escherichia coli Cause Urinary Tract Infections among Pregnant Women at Thi-Qar province, Iraq

The prevalence of antibiotic resistance in Enterobacteriaceae has increased sharply in recent years. Extendedspectrum ß-lactamase-producing Enterobacteriaceae include Escherichia coli has become especiallycommon. Although traditionally linked to risk factors such as prior hospitalization and antibiotic use, thesebacteria have become increasingly recognized in the community, especially as pathogens in urinary tractinfections (UTIs). In this study urine samples from 150pregnant women clinically diagnosed with UTIswere used for Gram staining, culture, API 20 E and singleplex PCR. Singleplex PCR was performed withprimers targeted to chuA and yjaA genes and anonymous DNA fragment TspE4C2 of E. coli. The positivesingleplex PCR products were identified by presence of 279 bp, 211 bp and 152 bp amplicons of chuA andyjaA genes and anonymous DNA fragment TspE4C2 for of E.coli. Conventional methods of Gram staining,culture and API 20E test showed positive result for E. coli in 35 (40%) out of 150 pregnant clinicallydiagnosed with urinary tract infection. PCR detected 24 (27.5%) out of the 35 (40%) samples that werepositive for E. coli. The majority of UTIs caused by spectrum ß-lactamase-producing Enterobacteriaceaeinclude E. coli was acquired in the community, so rapid, specific and sensitive molecular are urgently neededto better prevalence, prevent and treat these infections in Iraq

Antimicrobial Resistance and Virulence Characterization of E. Coli Isolated from Subclinical Mastitic Sheep and Goats

Mastitis is one of the most serious healthy and economic problems in all dairy sheep and goat flocks. Examination of 200 milk samples collected from apparently healthy sheep and goats using California mastitis test (CMT) showed that 94 (47%) were CMT positive. Standard methods for isolation and identification of E. coli could identify 19 strains. Five different serogroups were successfully identified among 19 E. coli strains, the serogroups were O1 (21.1%), O26 (21.1%), O114 (15.8%), O148 (15.8%) and finally O125 (10.4%), in addition to 3 untypable strains. The antimicrobial susceptibility results for erythromycin and cefoxtin revealed all E. coli isolates were resistant to erythromycin (100%) followed by high frequencies of resistance observed to cefoxtin 18 isolates (94.7%). Phylogenetic grouping of E. coli by triplex PCR using two genes (chuA and yjaA) and an anonymous DNA fragment tspE4C2 indicated that 5/6 were B2 group and 1/6 was A group. Detection of virulence genes of 6 E. coli. isolates showed that 2/6 harbored eaeA gene and none of 6 isolates harbored CFA/I gene. While, the mph A erythromycin resistance gene were present in 4/6.but 2/6 isolates harbored bla CTX cefoxitin resistance gene.

Distribution Pattern of EcoR Phylogenetic Groups Among Shiga Toxin-Producing and Enteropathogenic Escherichia coli Isolated From Healthy Goats

Background: Escherichia coli can be categorized into four major phylogenetic groups (A, B1, B2, and D) based on presence or absence of three markers including two genes (chuA and yjaA) and an anonymous DNA fragment designated TSPE4.C2. Also, these groups are divided into seven phylogenetic subgroups A0, A1, B1, B22, B23, D1, and D2. Objectives: This study aimed to determine the distribution pattern of phylogenetic groups in Shiga Toxin-Producing Escherichia coli (STEC) and Enteropathogenic Escherichia coli (EPEC) isolated from asymptomatic goats in Kerman city, Iran. Materials and Methods: Two hundred and fifty fecal samples were obtained from healthy goats. All isolates were subjected to detection of phylogenetic markers chuA, yjaA and DNA fragment TspE4.C2 and virulence genes stx1, stx2 and eae. Results: In summary, among all isolates phylo-group B1 was the most prevalent (57.6%) and other phylo-groups were A1 (20.4%), A0 (18.4%), D1 (2.8%), and B22 (0.8%). There was no isolate in B23 and D2 subgroups. Fifty samples (20%) possessed at least one of the tested virulencegenes : stx1 (12%), stx1/stx2 (4%), eae (2.8%), stx1/eae (0.8%), and stx2 (0.4%). Thus, 41 (16.4%) STEC, 7 (2.8%) EPEC, and 2 (0.8%) Enterohemorrhagic Escherichia coli (EHEC) strains were isolated and allocated into four phylogenetic subgroups A0 (16%), A1 (12%), B1 (68%), and D1 (4%). Conclusions: Based on 250 fecal samples obtained from goats in industrial slaughterhouse of Kerman City, goats may be a potential reservoir of STEC in Kerman and B1 followed by A are the most prevalent phylogenetic groups among STEC and non-STEC isolates in this study.

Identification of integrons and phylogenetic groups of drug-resistant Escherichia coli from broiler carcasses in China

The dissemination of drug-resistant Escherichia coli in poultry products is becoming a public concern, as it endangers food security and human health. It is very common for E. coli to exhibit drug resistance in the poultry industry in China due to the excessive use of antibiotics. However, few studies have examined the drug resistance endowed by integrons and integron-associated gene cassettes in different phylogenetic groups of E. coli isolated from broiler carcasses. In this study, 373 antibiotic-resistant E. coli strains were isolated from the surfaces or insides of broiler carcasses from a slaughterhouse in Shandong Province, China. According to phylogenetic assays of chuA, yjaA, and an anonymous DNA fragment, TSPE4-C2, these isolates belong to four phylogenetic groups (A, B1, B2, and D) and seven subgroups (A0, A1, B1, B21, B22, D1, and D2). Of the tested isolates, 95.71% (n=357) are multi-drug resistant, among which group B1 was predominant, accounting for 33.51% (n=125) of the tested isolates. A high percentage of the E. coli isolates were resistant to amoxicillin–clavulanic acid (99.20%, n=370), doxycycline (92.23%, n=344), sulfamethoxazole–trimethoprim (90.88%, n=339), ciprofloxacin, (64.61%, n=241), sulbactam–cefoperazone (51.21%, n=191), and amikacin (33.78%, n=126). Furthermore, among the 373 isolates, class 1 and 2 integrons were identified in 292 (78.28%) and 49 (13.14%) of the isolates, respectively, while no class 3 integrons were detected. The most prevalent gene cassette arrays were dfrA17–aadA5 and dfrA12–orfF–aadA2 in the variable region of class 1 integrons, while only one gene cassette array (dfrA1–sat2–aadA1) was detected in the variable region of class 2 integrons. Class 1 integrons were distributed in various physiological subtypes, whereas no predominant phylogenetic groups could be identified. The presence of class 2 integrons in the B21 subtype was significantly higher than in the other subtypes, and it coexisted with the class 1 integron. This study suggests that broiler products are potential sources of multi-drug resistant E. coli, and that resistance genes could be spread by lateral gene transfer.

Transferability of Sorghum Genic Microsatellite Markers to Peanut

Currently development of new marker types has shifted from anonymous DNA fragments to gene-based markers. Simple Sequence Repeats (SSRs) are useful DNA markers in plant genetic research including in peanut. However, de novo development of SSRs is expensive and time consuming. Gene-based DNA markers are transferable among related species owing to the conserved nature of genes. In this study transferability of sorghum EST-SSR (SbEST-SSR) markers to peanut was prospected. A set of 411 SbEST-SSR primer pairs were used to amplify peanut genomic DNA extracted from cultivated peanut where 39% of them successfully amplified. A comparison of amplification patterns between sorghum and peanut showed similar banding pattern with majority of transferable SbEST-SSRs. Among these transferable SSR markers, 14% have detected polymorphism among 4 resistant and 4 susceptible peanut lines for rust and late leaf spot diseases. These transferable markers will benefit peanut genome research by not only providing additional DNA markers for population genetic analyses, but also allowing comparative mapping to be possible between peanut and sorghum—a possible monocot-dicot comparison.

Molecular phylogeny of Escherichia coli isolated from clinical samples in Hilla, Iraq

Escherichia coli strains can be assigned to one of the main phylogenetic groups (A, B1, B2 and D). Strains of these groups differ in their phenotypic characteristics, including the ability to use certain sugars, antibiotic resistance profiles and growth rate-temperature relationships. A total of 45 E. coli isolates were obtained from different clinical samples by standard bacteriological methods. PCR was conducted to determine the phylogenetic grouping of these isolates by targeting two genes, chuA, yjaA and anonymous DNA fragment TspE4.C2. Three phylogeny genetic markers (chuA, yjaA and TspE4.C2) were used. Results found that the most isolates of E. coli belong to the phylogeny group A (44.4%) which includes: urine (3 samples), stool (8 samples), rectal (6 samples), and vagina (3 samples), followed by group B1 (22.2%) which included urine (4 samples), stool (2 samples), rectal (3 samples) and vagina (1 samples), group B2 (17.7%) which included urine (5 samples), and vagina (3 samples), while group D (15.5%) which included urine (3 samples), rectal (1 sample) and 3 vagina samples. Phylogeny pedigree was done according to the data recovered previously. This study explains that the distributions of E. coli isolates in phylogenetic groups (A, B1, B2 and D) varied depending on the characteristics of host population and also on the variation in bacterial sources. Key words : Phylogeny, Escherichia coli, polymerase chain reaction.

Application of Wavelet Packet Transform to detect genetic polymorphisms by the analysis of inter-Alu PCR patterns

BackgroundThe analysis of Inter-Alu PCR patterns obtained from human genomic DNA samples is a promising technique for a simultaneous analysis of many genomic loci flanked by Alu repetitive sequences in order to detect the presence of genetic polymorphisms. Inter-Alu PCR products may be separated and analyzed by capillary electrophoresis using an automatic sequencer that generates a complex pattern of peaks. We propose an algorithmic method based on the Haar-Walsh Wavelet Packet Transformation (WPT) for an efficient detection of fingerprint-type patterns generated by PCR-based methodologies. We have tested our algorithmic approach on inter-Alu patterns obtained from the genomic DNA of three couples of monozygotic twins, expecting that the inter-Alu patterns of each twins couple will show differences due to unavoidable experimental variability. On the contrary the differences among samples of different twins are supposed to originate from genetic variability. Our goal is to automatically detect regions in the inter-Alu pattern likely associated to the presence of genetic polymorphisms.ResultsWe show that the WPT algorithm provides a reliable tool to identify sample to sample differences in complex peak patterns, reducing the possible errors and limits associated to a subjective evaluation. The redundant decomposition of the WPT algorithm allows for a procedure of best basis selection which maximizes the pattern differences at the lowest possible scale. Our analysis points out few classifying signal regions that could indicate the presence of possible genetic polymorphisms.ConclusionsThe WPT algorithm based on the Haar-Walsh wavelet is an efficient tool for a non-supervised pattern classification of inter-ALU signals provided by a genetic analyzer, even if it was not possible to estimate the power and false positive rate due to the lacking of a suitable data base. The identification of non-reproducible peaks is usually accomplished comparing different experimental replicates of each sample. Moreover, we remark that, albeit we developed and optimized an algorithm able to analyze patterns obtained through inter-Alu PCR, the method is theoretically applicable to whatever fingerprint-type pattern obtained analyzing anonymous DNA fragments through capillary electrophoresis, and it could be usefully applied on a wide range of fingerprint-type methodologies.

A combination of techniques proves useful in the development of nuclear markers in the newt genus Triturus

To increase the number of markers available for study of phylogeny and phylogeography in the newt genus Triturus, we developed and tested 59 primer pairs using three different techniques. Primers were obtained from published sources, by designing exon-primed intron-crossing primers and from randomly cloned anonymous nuclear DNA fragments. Successful polymerase chain reaction products were cloned and sequenced. Five fragments were successfully amplified and sequenced for six species of Triturus: intron 7 of the β-fibrinogen gene (βfibint7), third intron of the calreticulin gene (CalintC), the 11th intron of the α-subunit of the platelet derived growth factor receptor (PDGFRα) and two anonymous markers (Cri1 and Cri4). The average percentage species divergence across all the markers is low (c. 3%), compared to what has been found in mitochondrial DNA (25-30%).

The effect of sequencing errors on metagenomic gene prediction

BackgroundGene prediction is an essential step in the annotation of metagenomic sequencing reads. Since most metagenomic reads cannot be assembled into long contigs, specialized statistical gene prediction tools have been developed for short and anonymous DNA fragments, e.g. MetaGeneAnnotator and Orphelia. While conventional gene prediction methods have been subject to a benchmark study on real sequencing reads with typical errors, such a comparison has not been conducted for specialized tools, yet. Their gene prediction accuracy was mostly measured on error free DNA fragments.ResultsIn this study, Sanger and pyrosequencing reads were simulated on the basis of models that take all types of sequencing errors into account. All metagenomic gene prediction tools showed decreasing accuracy with increasing sequencing error rates. Performance results on an established metagenomic benchmark dataset are also reported. In addition, we demonstrate that ESTScan, a tool for sequencing error compensation in eukaryotic expressed sequence tags, outperforms some metagenomic gene prediction tools on reads with high error rates although it was not designed for the task at hand.ConclusionThis study fills an important gap in metagenomic gene prediction research. Specialized methods are evaluated and compared with respect to sequencing error robustness. Results indicate that the integration of error-compensating methods into metagenomic gene prediction tools would be beneficial to improve metagenome annotation quality.

Assigning Escherichia coli strains to phylogenetic groups: multi‐locus sequence typing versus the PCR triplex method

It is well recognized that Escherichia coli consists of a number of distinct phylo-groups and that strains of the different phylo-groups vary in their ecological niches, life-history characteristics and propensity to cause disease. Consequently, much can be learnt by assigning a strain of E. coli to one of the recognized phylo-groups. A triplex PCR-based method that enables strains of E. coli to be assigned to a phylo-group using a dichotomous key approach based on the presence or absence of two genes (chuA and yjaA) and an anonymous DNA fragment (TSPE4.C2) has been developed. However, the accuracy with which this method assigns strains to their correct phylo-group has not been adequately evaluated. Consequently, 662 strains of E. coli were characterized using a multi-locus sequence typing approach. Unsupervised population assignment algorithms were used to assign strains to phylo-groups based on the multi-locus sequence typing data. The analyses revealed that 85-90% of E. coli strains can be assigned to a phylo-group and that 80-85% of the phylo-group memberships assigned using the Clermont method are correct. However, the accuracy with which strains are assigned to the correct phylo-group depends on their Clermont genotype. For example, strains yielding a Clermont genotype consistent with phylo-groups B1 and B2 are assigned correctly 95% of the time. Strains failing to yield any PCR products using the Clermont method are seldom members of phylo-group A and strains with such a genotype should not be assigned to a phylo-group.

Refinement of localization of the human genes for myeloperoxidase (MPO), protein kinase C, a polypeptide, PRKCA, and the DNA fragment D17S21 on chromosome 17q

The reciprocal translocation t(2;17) (q21.2; q23.2) was used to map the human genes for myeloperoxidase (MPO), protein kinase C, alpha polypeptide, PRKCA, and the anonymous DNA fragment D17S21 more precisely on human chromosome 17q. MPO and PRKCA remain on the der(17) chromosome and therefore are assigned to 17(q21.3-q23.2) and 17(q22-q23.2), respectively. D17S21 is translocated to chromosome der(2) and by this is assigned to 17(q23.3-q24).

Human gene mapping.

It is now possible to map the human genome completely with a set of closely linked markers. Over 500 coding genes have been cloned and localized, as have approximately 2000 anonymous DNA fragments, most of which recognize two-allele polymorphisms that are caused by single base changes which alter the recognition site for a restriction enzyme (restriction fragment length polymorphisms). Most human chromosomes have been mapped, with markers in defined order placed approximately 10 map units apart. Chromosomes X and 21 are particularly well mapped, with over 200 probes ordered on X. The strategy during the next few years will encompass moving from a linkage map to a set of overlapping cosmid or phage clones, and finally to a complete sequence of regions of chromosomes and entire chromosomes. A complete sequence of the human genome should transform our understanding of development, the control of gene expression, and the parameters of genetic disease.

Rapid and simple determination of the Escherichia coli phylogenetic group.

Phylogenetic analysis has shown that Escherichia coli is composed of four main phylogenetic groups (A, B1, B2, and D) and that virulent extra-intestinal strains mainly belong to groups B2 and D. Actually, phylogenetic groups can be determined by multilocus enzyme electrophoresis or ribotyping, both of which are complex, time-consuming techniques. We describe a simple and rapid phylogenetic grouping technique based on triplex PCR. The method, which uses a combination of two genes (chuA and yjaA) and an anonymous DNA fragment, was tested with 230 strains and showed excellent correlation with reference methods.

Genealogical relationships among cultivated avocado as revealed through RFLP analyses

Anonymous DNA fragments from the genome of cultivated avocado (Persea americana Mill.) were cloned into a plasmid vector and used to screen a total of 36 cultivars. There is a high level of polymorphism among cultivars allowing all cultivars to be assigned a unique genotype based on 14 genetic loci. A cluster analysis of genetic similarities among cultivars revealed three major clusters that correspond to the three major racial groupings of cultivated avocado. Additional clusters appear to reflect cultivars derived through interracial hybridization.

Comparative Mapping in thebeige–satinRegion of Mouse Chromosome 13

The proximal end of mouse chromosome (Chr) 13 contains regions conserved on human chromosomes 1q42–q44, 6p23–p21, and 7p22–p13. This region also contains mutations that may be models for human disease, includingbeige(human Chediak–Higashi syndrome). An interspecific backcross of SB/Le andMus spretusmice was used to generate a molecular genetic linkage map of mouse chromosome 13 with an emphasis on the proximal region includingbeige(bg) andsatin(sa). This map provides the gene order of the two phenotypic markersbgandsarelative to restriction fragment length polymorphisms and simple sequence length polymorphisms in 131 backcross animals. In parallel, we have created a physical map of the region usingNidogen(Nid) as a molecular starting point for cloning a YAC contig that was used to identify thebeigegene. The physical map provides the fine-structure order of genes and anonymous DNA fragments that was not resolved by the genetic linkage mapping. The results show that thebgregion of mouse Chr 13 is highly conserved on human Chr 1q42–q44 and provide a starting point for a complete functional analysis of the entirebg–sainterval.

Amplification of anonymous DNA fragments using pairs of long primers generates reproducible DNA fingerprints that are sensitive to genetic variation.

The reproducibility and potential applications of anonymous amplification protocols can be improved by using pairs of primers, each of 18 to 24 bases, to replace the single 8 to 10 base primers normally used in randomly amplified polymorphic DNA (RAPD) or DNA amplification fingerprinting (DAF) methods. Amplification using large primer pairs (LP-RAPD) generates 5 to 30 bands that can be resolved on standard agarose gels. Complex fingerprints can be readily generated from viruses, bacteria, fungi, plants, invertebrates and vertebrates. We also present evidence that a number of polymerase chain reaction (PCR) methods, including those based on the use of enterobacterial repetitive intergenic consensus (ERIC-PCR) or microsatellite primed (MP-PCR) sequence, may in essence operate by the same mechanism as LP-RAPD. Using standard LP-RAPD protocols, reproducible fingerprints can be generated from a single specimen using different thermocyclers, regardless of the mechanism used for thermocycling (air-cooled, Peltier effect, or robotic arm). LP-RAPD is sensitive to intraspecific and interspecific genetic variation, demonstrated here by analysis of mites and apple cultivars. Approximately 50% of LP-RAPD products are expected to have different primers at either end. Polymorphic bands with this arrangement can be recovered from the gel and directly sequenced using the LP-RAPD primers themselves. The efficiency of sequencing is improved by the length of the LP-RAPD primers. This method has the potential to allow the production of allele-specific species markers in less than two days.

Isolation of CpG Island Fragments: a Putative Promoter Region of the Human Prostacyclin Synthase Gene.

An anonymous human DNA fragment R16-2, which was isolated as a possible CpG island fragment by the segregation of partly melted molecules method (SPM), was found to contain a putative promoter region of the prostacyclin synthase gene. Based on this data, it is suggested that combined use of the SPM technique and the database match enables a fine characterization of the regulatory region of many genes.

Differential cloning using in-gel competitive reassociation.

We describe the principle and actual processes of a differential cloning procedure designed for cloning of anonymous restriction DNA fragments whose molecular sizes differ between two genomic DNA preparations from higher organisms as a result of DNA rearrangement, polymorphism, etc. The procedure, which was extensively modified from the original one and still employs in-gel competitive reassociation (IGCR) as the basic principle, aims for cloning of DNA fragments which exist in one copy or less per mammalian genome. The modified procedure consists of dissociation and reassociation of biotinylated restriction digests of target DNA fragments (from which clones are to be isolated) in the presence of a large excess of reference (competitor) DNA in gel after electrophoresis, which is followed by absorption of the target DNA fragments to streptavidin-coated tubes and solid-phase polymerase chain reaction. After repeating these steps we attained substantial enrichment of altered DNA fragments which were originally present in one copy or less per complex mammalian genome.

A Differential Cloning Procedure of Complex Genomic DNA Fragments

We have developed a differential cloning procedure designed for cloning of anonymous restriction DNA fragments whose molecular sizes differ between two genomic DNA preparations from higher organisms. The procedure, which was extensively revised from the original one, consists of several steps as summarized below. (i) Digestion of two DNA preparations (target and reference DNA) with the same restriction enzyme (4 base cutter). (ii) Biotinylation of target DNA and conversion of reference DNA to nonamplifiable form by terminal dephosphorylation. (iii) Electrophoresis of the two DNA preparations through a synthetic gel with a large excess of reference DNA as a competitor. (iv) In-gel alkaline dissociation of DNA, followed by reassociation (in-gel competitive reassociation). (v) Elution of DNA from the gel and PCR after adapter ligation and adsorption of DNA onto streptavidin-coated matrix. By repeating these steps, we attained substantial enrichment (∼10,000-fold) of DNA fragments which were originally present at one copy or less per complex mammalian genome. The details of the procedure and its unique characteristics in cloning of altered genomic DNA fragments, particularly from mammalian genome, are discussed.

Gene mapping from a bovine 1;29 DNA library prepared with chromosome microdissection.

Bovine gene mapping is progressing rapidly using syntenic group mapping based on somatic cell hybrids and linkage, and to a lesser extent on in situ hybridization. Single chromosome DNA libraries are a logical next step, and this was, therefore, the aim of our laboratory. Since we have access to several cattle with t(1;29) and this chromosome is readily distinguishable, we chose this as our first target--recognizing that we would not produce a "single" chromosome library in the strict sense because two autosomes are represented. We utilized an inverted microscope and a micromanipulator fitted with glass instruments pulled specifically to dissect off approximately 100 t(1;29) chromosomes per microdrop. A glass chamber made to accommodate a hanging drop was used to extract the DNA under a dissecting microscope. The DNA was then cleaved with EcoRI and inserted in lambda gtwes arms. Host cells were then infected with these phage and positive clones obtained. The first clone, isolated from this library by hybridization with a human collagen 6A1 cDNA, was mapped by in situ hybridization to bovine Chromosome some (Chr) 1q12-q14, near the centromere. The second clone, an anonymous DNA fragment (D1S11), was mapped to 1q43-q46, near the terminal end.