The Novel HLA-C*03:712 Allele, Identified by Sanger Dideoxy Nucleotide Sequencing in a Chinese Individual.
The HLA-C*03:712 allele differs from HLA-C*03:03:01:01 by one nucleotide substitution in codon 61 in exon 2.
Humans↗
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Results for “sequence based typing”
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Pseudomonas aeruginosa serotype O12 outbreak studied by arbitrary primer PCR.
A total of 16 colonizing and infecting ofloxacin-resistant Pseudomonas aeruginosa strains and two strains isolated from ventilation equipment fluids, all with similar colonial morphologies and with minor but distinct susceptibility differences, were suspected of belonging to a single outbreak and were studied by arbitrary primer (AP) PCR. Thirteen nonrelated strains were included to evaluate the discriminatory capacity of the technique. AP PCR fingerprinting was compared with serotyping, phage typing, and antibiotic susceptibility testing. AP PCR was performed independently with three different primers. The different AP PCR typing systems yielded almost identical patterns for the epidemic strains and enabled us to differentiate most of the nonrelated strains from each other and from the outbreak strains. The combination of AP PCR typing and the phenotyping techniques that we used enabled us to conclude that an outbreak was occurring. In general, the typeability of AP PCR was greater than those of phage typing and serotyping, while the discriminatory powers of the three methods were comparable.
Bacterial Typing Techniques↗
Risk of cross-colonization and infection by Pseudomonas aeruginosa in a holiday camp for cystic fibrosis patients.
The risk of cross-colonization and subsequent infection by Pseudomonas aeruginosa in holiday camps for cystic fibrosis patients was studied in 91 children by culturing sputum at their arrival, at their departure, 2 months later, and at regular intervals thereafter. The isolated strains were subjected to serotyping, phage typing, pyocin typing, and genotyping by random amplified polymorphic DNA fingerprinting-PCR. It was concluded from random amplified polymorphic DNA fingerprinting-PCR typing that the Pseudomonas flora was not constant in most children. Some children harbored one genotype, whereas some harbored two or more different genotypes simultaneously. Most culture-positive children easily acquired a strain of another genotype which replaced the former one or coexisted with the original one. The incidence of sputum conversion was 7.7% in previously negative children; the incidence of permanent colonization and infection was 1.9%. This risk was comparable with that observed in the community. We conclude that the risk of cross-infection is trivial compared with the obvious joy and social benefit derived from a holiday camp.
Adolescent↗
Epidemiology of Salmonella enterica serotype enteritidis in southern Italy in the period January 1995-June 1998.
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Animals↗
[The prevalence of methicillin-resistant Staphylococcus aureus phagotype 95 in the Hospitales Vall d'Hebron of Barcelona].
BACKGROUND: In our hospital endemic methicillin resistant Staphylococcus aureus (MRSA) has been documented since 1971, with epidemic and interepidemic periods. During these years phage groups I, I-III, and non-typable were found by the international set of phages Phage group 95 (F95) was unusual between 1986 (when phage typing was first available) and 1991, with prevalence of 5.2% (mean), and 100% of sensibility to methicillin. In November 1991 appeared the first MRSA F95 strain, and its prevalence has been increasing until 1997. MATERIAL AND METHODS: We have studied 133 strains of MRSA F95 isolated from 87 patients, 39 of them hospitalized in the General Hospital (HG), 38 in Traumatology Hospital (HT) and 8 in the Children's Hospital (HI). Two of these patients had successive stancies in HG and HT. Antimicrobial susceptibility was determined by the disk diffusion method and microdilution to check oxacillin resistance. Moreover these method we have maked: detection of mecA, phage typing with the international set of phages and study of the PGFE patterns by digestion of chromosomic DNA with Smal. RESULTS: The percentage of methicillin resistance in F95 strains was increased since the appear of the first strain between 8.3% in 1991 to a maximum of 76.9% in 1995, we had a descens to 13.7% in 1996 but 1997 can back to augment it to 72.5%. MICs for oxacillin of these strains were low (< or = 64 mg/l to 87.4% of strains), and all of them were mecA positive, 78.1% of them were resistant to macrolides, 96.5% to tobramycin and 84.9% to quinolones, but only 10.5% to gentamicin, 4.7% were resistant to cotrimoxazol, 1.2% to fosfomycin and 2.5% to rifampin. All of them were sensible to doxycycline, and vancomycin. The pulse field gel electrophoresis showed 7 restriction patterns in MRSA F95, 73.8% of strains correspond to one of them (B), spreading from the spinal cord injury unit and prevalent in HT; and 10.8% to another (C), the first that appear, spreading from the neurosurgical unit and with high prevalence in HG. 6.9% has pattern J a B subtype that appear in broth HG and HT. Pattern E is prevalent in HI it was spread from neonatology unit. CONCLUSIONS: The emergence in a Center with endemic resistance of new strains of MRSA, not all of them of the same clone, with characteristic resistance pattern to antibiotics and in convivence with other phage groups is one demonstration of genetic variability of SAMR in our entorn.
Bacteriophage Typing↗
[Current status of bacterial taxonomy and its importance for medical microbiology].
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Bacteria↗
Repetitive sequence-based PCR versus pulsed-field gel electrophoresis for typing of Enterococcus faecalis at the subspecies level.
Repetitive sequence-based PCR was compared to pulsed-field gel electrophoresis (PFGE) for the ability to discriminate Enterococcus faecalis isolates at the subspecies level. The BOXA2R primer, derived from repetitive sequences in Streptococcus pneumoniae, was applied to 41 isolates of E. faecalis collected from various sources. The REP1R-Dt and REP2-Dt primers, derived from the gram-negative repetitive extragenic palindromic element, were also applied to 18 selected isolates. Of the 41 isolates examined, 7 were beta-lactamase producing and 8 were vancomycin resistant. By PFGE, 17 isolates had distinct patterns; the other 24 were classified into eight different clonal groups. By PCR using the BOXA2R primer, 16 isolates generated distinct patterns; the other 25 were classified into nine different clonal groups. There were only minor differences in the PCR results obtained by using the BOXA2R primer and the REP1R-Dt and REP2-Dt primers. Two isolates among vancomycin-resistant enterococci from the greater Houston, Tex., area were related by PFGE, distinct by PCR with the BOXA2R primer, and related by PCR with the REP1R-Dt and REP2-Dt primers. Clonal relationships among the remaining 39 isolates were similar by both PFGE and PCR. PCR reliably discriminated all epidemiologically unrelated isolates. Although PCR is less time consuming than PFGE, PCR results were more difficult to interpret than PFGE results, perhaps because fewer bands were generated by PCR than by PFGE and some PCR products were inconsistently seen.
Bacterial Typing Techniques↗
Characterisation of group A streptococcal isolates from tropical Australia with high prevalence of rheumatic fever: probing for signature sequences to identify members of the family of serotype 5.
The prevalence of rheumatic heart disease (RHD) in Northern Territory Aboriginal communities is high, but there is a low isolation rate of historically rheumatic fever associated M types (such as M5) of group A streptococci (GAS). Many isolates are M non-typable (MNT). Serology suggests that the population is exposed to M5-like isolates; some RHD patients having high IgM or IgG titres to two M5 B-repeat region peptide epitopes, B1 (KQQESK) and B4 (EQKSKQ). To identify relatives of M5 in our collection of GAS, oligonucleotide probes to the B1 and B4-repeat regions shared by M5 and a local M5-like isolate, were used to screen 101 isolates for the presence of signature sequences. In all, 28% of the tropical Australian isolates contained the signature sequences, identifying members of the M5 family. The 5' region of the genes for M proteins from three members of the M5 family fell into two sequence types. Hybridisation to probes based on these sequences suggested that among tropical Australian isolates there are at least three distinct sequence types that contained the M5 signature sequences. These results suggest that a considerable number of M5 family GAS are circulating in tropical Australia.
Amino Acid Sequence↗
Base-pair resolution conservation data improves cell type specific sequence-to-expression prediction.
MOTIVATION: Genomic sequence-to-activity models can decipher gene regulatory mechanisms and predict the functional impact of regulatory variants. However, current models struggle to integrate information from sequences outside promoters, especially information from cell type specific regulatory elements. RESULTS: Here, we propose incorporating base-pair resolution evolutionary conservation data into genomic sequence-to-expression predictors. We explore two training strategies-training from scratch or fine-tuning an existing sequence-only model with additional conservation input. We find that in both cases, base-pair resolution conservation data improves cell type specific sequence-to-expression prediction, with training from scratch yielding the greatest benefit. The improvement in cell type specific expression prediction can be attributed in part to the fact that models trained on sequence and conservation data learn to better recognize cell type specific regulatory elements than models trained on sequence alone. AVAILABILITY: Code is available at https://github.com/ni-lab/basenji-phyloP.
Conserved Sequence↗
NetOglyc: prediction of mucin type O-glycosylation sites based on sequence context and surface accessibility.
The specificities of the UDP-GalNAc:polypeptide Nacetylgalactosaminyltransferases which link the carbohydrate GalNAc to the side-chain of certain serine and threonine residues in mucin type glycoproteins, are presently unknown. The specificity seems to be modulated by sequence context, secondary structure and surface accessibility. The sequence context of glycosylated threonines was found to differ from that of serine, and the sites were found to cluster. Non-clustered sites had a sequence context different from that of clustered sites. Charged residues were disfavoured at position -1 and +3. A jury of artificial neural networks was trained to recognize the sequence context and surface accessibility of 299 known and verified mucin type O-glycosylation sites extracted from O-GLYCBASE. The cross-validated NetOglyc network system correctly found 83% of the glycosylated and 90% of the non-glycosylated serine and threonine residues in independent test sets, thus proving more accurate than matrix statistics and vector projection methods. Predictions of O-glycosylation sites in the envelope glycoprotein gp120 from the primate lentiviruses HIV-1, HIV-2 and SIV are presented. The most conserved O-glycosylation signals in these evolutionary-related glycoproteins were found in their first hypervariable loop, V1. However, the strain variation for HIV-1 gp120 was significant. A computer server, available through WWW or E-mail, has been developed for prediction of mucin type O-glycosylation sites in proteins based on the amino acid sequence. The server addresses are http://www.cbs.dtu.dk/services/NetOGlyc/ and netOglyc@cbs.dtu.dk.
Algorithms↗
Mutations that alter an Arg-Gly-Asp (RGD) sequence in the adenovirus type 2 penton base protein abolish its cell-rounding activity and delay virus reproduction in flat cells.
The adenovirus penton base protein has a cell rounding activity and may lyse endosomes during virus entry into the cytoplasm. We found that penton base that was expressed in Escherichia coli also caused cell rounding and that cells adhered to polystyrene wells that were coated with the protein. Mutant analysis showed that both properties required an Arg-Gly-Asp (RGD) sequence at residues 340 to 342 of penton base. In flat adherent cells, virus mutants with amino acid substitutions in the RGD sequence were delayed in virus reproduction and in the onset of viral DNA synthesis. In nonadherent or poorly spread cells, the kinetics of mutant virus reproduction were similar to those of wild-type adenovirus type 2. Expression of the mutant phenotype exclusively in the flat cells that we tested supports a model in which penton base interacts with an RGD-directed cell adhesion molecule during adenovirus uptake or uncoating.
Adenoviruses, Human↗
High-resolution HLA-DPB typing based upon computerized analysis of data obtained by fluorescent sequencing of the amplified polymorphic exon 2.
To differentiate 32 HLA-DPB alleles, conventional techniques such as serology and cellular typing are inadequate for high-resolution DPB typing. The most refined DNA typing until now is SSO typing and new selected oligonucleotides can be added to this system to distinguish new allele sequences. DNA sequencing, however, reveals directly the sequence information of all polymorphic HVRs and has the advantage of being independent from exon polymorphisms. We have developed a new DNA-based typing approach that is rapid, fully automated, and therefore suitable for routine typing. The system is based upon direct sequencing of amplified DNA with fluorescent-labeled primers. The designation of alleles is obtained by a comparison of all polymorphic positions in the determined sequence with all known allele sequences retained in a database along with their heterozygous combinations. Sequence data at both constant and polymorphic positions are used for quality control. In this study, the typing results of a panel of 91 previous SSO-typed DNA samples are described. After comparison with the SSO-typing results, we conclude that with this SBT system allele assignment is reliable. The method is easy to perform since both sequencing and assignment are automated. Furthermore, the system is easily applicable to other gene systems.
Base Sequence↗
In vitro evolution used to define a protein recognition site within a large RNA domain.
A minimum of 460 nucleotides of 16S ribosomal RNA are needed to fold the target site for E. coli ribosomal protein S4, although a much smaller region within this large domain is protected from chemical reagents by the protein. Starting with a 531-nucleotide tRNA fragment, cycles of mutagenesis, selection with S4, and amplification ('in vitro evolution') were used to obtain a pool of 30 RNA sequences selected for S4 recognition but approximately 30% different from wild type. Numerous compensatory base pair changes have largely preserved the same secondary structure among these RNAs as found in wild-type sequences. A 20-base deletion and a single nucleotide insertion are among several unusual features found in most of the selected sequences and also prevalent among other prokaryotic rRNAs. Most of the compensatory base changes and selected features are located outside of the region protected by S4 from chemical reagents. It was unexpected that S4 would select for RNA structures throughout such a large domain; the selected features are probably contributing indirectly to S4 recognition by promoting correct tertiary folding of the region actually contacted by S4. The role of S4 may be to stabilize this domain (nearly one-third of the 16S rRNA) in its proper conformation for ribosome function.
Base Sequence↗
The importance of base pairing in the penultimate stem of Escherichia coli 16S rRNA for ribosomal subunit association.
The influence of base pairing in the penultimate stem of Escherichia coli 16S rRNA (defined as nt 1409-1491) on ribosome function has been addressed by the construction of mutations in this region of rRNA. Two sets of mutations were made on either side of a structurally conserved region in the penultimate stem that disrupted base pairing, while a third set of mutations replaced the wild-type sequence with other base pair combinations. The effects of these mutations were analyzed in vivo and in vitro . The mutations that disrupted base pairing caused significant increases in cell doubling times as well as a severe subunit association defect and a modest increase in frame shifting and stop codon read-through. Restoration of base pairing restored wild-type growth rates, decoding and subunit association, indicating that base pairing in this region is essential for proper ribosome function.
Base Sequence↗
Typing of Mycoplasma pneumoniae by nucleic acid sequence-based amplification, NASBA.
Nucleic acid sequence-based amplification, NASBA, is an isothermal amplification technique for nucleic acids and was used for typing a collection of 24 Mycoplasma pneumoniae strains. A set of primers was chosen from the 16S rRNA sequence alignment of Mycoplasma species. The nucleotide sequences of the (-)RNA amplicons were determined for M. pneumoniae strains M15/83 (type 1) and FH (type 2), and revealed a one-point difference at the 16S rRNA level between the two types. Based on this result, two type-specific probes were constructed. The probes were hybridized in solution with the amplified nucleic acids of 24 M. pneumoniae strains in an enzyme-linked gel assay (ELGA). The results obtained by NASBA-based typing are in agreement with the classification of the 24 M. pneumoniae strains into two types by other typing methods, confirming the reliability of this technique.
Bacterial Typing Techniques↗
Analysis of clonal relationships among isolates of Shigella sonnei by different molecular typing methods.
Shigella sonnei is a major cause of diarrheal disease in developed as well as in developing countries. Epidemiologic studies of this organism have been limited by the lack of a simple and effective method for comparing strains. In this study, we have compared different molecular typing methods, i.e., plasmid profile analysis, restriction endonuclease analysis of plasmids, rRNA gene restriction analysis (ribotyping), pulsed-field gel electrophoresis (PFGE), and enterobacterial repetitive intergenic consensus (ERIC) sequence-based PCR (ERIC-PCR) for typing 20 clinical isolates of S. sonnei collected from six incidents of infection. PFGE and ERIC-PCR fingerprintings had the highest discriminatory power for discrimination of epidemiologically related isolates from epidemiologically unrelated strains of S. sonnei, and both gave seven distinct strain types among these isolates and the type strain of the species. Plasmid study and ribotyping produced only six and typing techniques demonstrated two distinct patterns, respectively, among these strains. All of these molecular an identical fingerprint for eight temporally related sporadic isolates. It is possible that these temporally related isolates belonged to a single bacterial clone and circulated obscurely through the community. Our results indicate that the ERIC-PCR technique represents a rapid and simple means for typing S. sonnei with a level of discrimination equivalent to that of PFGE but greater than those of plasmid profile analysis, restriction endonuclease analysis of plasmids, and ribotyping.
Bacterial Typing Techniques↗
Measurement of mRNA for E-selectin, VCAM-1 and ICAM-1 by reverse transcription and the polymerase chain reaction.
Stimulation of cultured human umbilical vein endothelial cells by cytokines such as interleukin-1 and tumour necrosis factor induces de novo synthesis and expression of the adhesion molecules E-selectin, vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1). In general, alterations in cell surface expression of these molecules are known to be related to increased gene transcription and altered levels of mRNA. The extension of these observations to the study of inflammatory processes in different human organs necessitates the development of techniques for the quantification of mRNA in small tissue samples. Here we present a method for the quantification of mRNA for E-selectin, VCAM-1 and ICAM-1 using reverse transcription and the polymerase chain reaction (RT-PCR). For each molecule of interest a mutant RNA was synthesised consisting of the wild-type sequence deleted of 15-20 bases. The mutant and wild-type RNA sequences are recognised by the same primers, and can therefore be amplified competitively in the same tube by RT-PCR. As the mutant and wild-type RNAs compete for the primers, the amount of wild-type RNA can be determined by the size of the dominant product that results after addition of known quantities of mutant RNA. Using this detection and quantification method we have examined the dose dependency and time course of mRNA accumulation following TNF-alpha stimulation of HUVEC. Similar time-courses of E-selectin, ICAM-1 and VCAM-1 mRNA accumulation were observed by competitive RT-PCR as by laser densitometry of Northern blots. Finally we were able to show that the technique could measure changes in levels of mRNA for these three molecules in human skin biopsies taken at different times during the development of a delayed hypersensitivity response to tuberculin purified protein derivative. This technique should be useful for the study of adhesion molecule mRNA in small tissue culture samples and in biopsies.
Base Sequence↗
Suitability of repetitive-DNA-sequence-based PCR fingerprinting for characterizing epidemic isolates of Salmonella enterica serovar Saintpaul.
Three molecular typing methods, repetitive-sequence-based PCR (rep-PCR) fingerprinting, plasmid profiling, and arbitrarily primed PCR fingerprinting, were used to characterize isolates of Salmonella enterica serovar Saintpaul. Most of the isolates were obtained from epidemic human cases of food-borne salmonellosis, together with some from the food material suspected to be the source of infection, and a few were obtained from other cases apparently not related to the epidemic. All three methods adequately discriminated the epidemic strain from other strains of the serovar. In addition several isolates from human cases which are not identical to the epidemic strain were found. These isolates therefore must have been responsible for some sporadic infections, which were only temporally related to the epidemic. These strains showed a high degree of similarity to a strain isolated from a turkey. rep-PCR fingerprinting with REP-Dt primers and primer ERIC1R, applicable even to crude cell lysates, offers an attractive choice as a primary method for the discrimination of various Salmonella serotypes as well as isolates within serotype Saintpaul.
Bacterial Typing Techniques↗