Showing papers on "Typing published in 1993"

HLA‐DQB1 and ‐DQA1 typing by PCR amplification with sequence‐specific primers (PCR‐SSP) in 2 hours

Abstract: In the present study PCR primers were designed for detecting all phenotypically expressed DQB1 and DQA1 allelic variability, 19 and 10 alleles, respectively, by PCR amplification with sequence-specific primers (PCR-SSP). For DQB1 typing, each sample was amplified by a first set of 14 PCR primer pairs, followed in some cases by two to six additional PCR reactions. The first 14 primer pairs allowed the identification/separation of all but a few of the recently described DQB1 alleles: DQB1*0504, DQB1*0605, DQB1*0606 and DQB1*0607 would not be identified; DQB1*0603 and DQB1*0608; and DQB1*0301 and DQB1*0304, respectively, would not be distinguished. Therefore an additional set of eight DQB1 primer pairs was used for a complete DQB1 typing, including all homozygous and heterozygous combinations. For DQA1 typing, 12 PCR reactions were performed per sample, 10 for detecting variability within the second exon and two for identifying first exon polymorphism. All homozygous and heterzoygous combinations of DQA1 alleles could be resolved by these primer pairs. In addition, four primer mixes were designed for determining codon 57 of the HLA-DQB1 gene. Thirty cell lines and 120 individuals were investigated by the DQB1 and DQA1 PCR-SSP technique, as well as with the HLA-DQ beta 57 primers. The concordance between PCR-SSP typing and assigning DQB1 and DQA1 alleles from TaqI DRB-DQA-DQB RFLP analysis was 100%. The reproducibility was 100% in 30 samples investigated on two separate occasions. Amplification patterns, investigated in 15 nuclear families, segregated according to dominant Mendelian inheritance. DQB1 and DQA1 PCR-SSP typing can be performed in 2 hours, including DNA extraction, PCR amplification and post-amplification processing. The method is technically simple and the typings are easy to interpret. The cost for typing one individual is low and is independent of the number of samples analyzed simultaneously, i.e. the technique is well-suited for routine clinical use.

Flagellin gene typing of Campylobacter jejuni by restriction fragment length polymorphism analysis.

Abstract: We developed and studied a molecular typing approach for Campylobacter spp. with restriction fragment length polymorphism (RFLP) analysis of the flagellin gene flaA in C. jejuni. Using polymerase chain reaction, we amplified the flaA gene from strains comprising different HL:O serotypes by using a primer set directed at the conserved 5' and 3' flaA gene sequence to generate a 1.7-kb amplicon. The amplicon was further digested with the restriction enzyme DdeI, and the fragments generated were analyzed by agarose gel electrophoresis. In 43 non-outbreak strains of six common HL serotypes (HL 1, 2, 4, 5, 9, and 36) in the United States, 18 RFLP patterns were observed. In U.S. outbreak strains previously studied by 10 other typing methods, flaA typing correlated with the HL serotype within each outbreak, and six additional flaA types were identified. Our results suggest that RFLP analysis of the flaA gene from Campylobacter spp. has sufficient discrimination to be useful as a practical typing method for clinical and epidemiologic investigations.

Typing of pneumococci by using 12 pooled antisera.

Abstract: A new simplified chessboard system for typing of Streptococcus pneumoniae is described. It is intended for typing or grouping of 90 to 95% of the pneumococcal strains most commonly isolated from blood or cerebrospinal fluid and is based on 12 pooled diagnostic antisera, each reacting with 7 to 11 single types, together covering the 23 different vaccine-related types as well as 25 other cross-reacting types. Worldwide surveillance of the type distribution is important in order to ensure an optimal formulation of pneumococcal polysaccharide vaccines and, in the future, of polysaccharide-protein conjugate vaccines. The simplified typing system described in this paper makes it easier to carry out surveillance in other than specialized reference laboratories. Finally, it takes advantage of the fact that some types cause disease more often in children--as opposed to adults--than do others.

PCR-amplification and detection of the human D1S80 VNTR locus Amplification conditions, population genetics and application in forensic analysis

Abstract: A series of experiments has been performed to evaluate amplification and typing of the D1S80 VNTR locus. The validation study that has been carried out showed that correct D1S80 typing results can be obtained when a defined amplification protocol and a high-resolution polyacrylamide gel electrophoresis method are used. The use of the Chelex extraction protocol has substantially reduced the processing time. DNA-extraction, amplification and subsequent typing can be performed in one day. The discrimination power of this locus is 0.94 in a Dutch Caucasian population sample. The system is extremely sensitive: 0.1 ng of genomic DNA gave a correct typing result. The test could also detect the correct genotypes in mixed samples containing DNA from different individuals. Even if the major type was in a 20-fold excess, the minority type could still be amplified and typed correctly. We have found no deviation from Hardy-Weinberg equilibrium in a Dutch Caucasian population sample. Evidence for the somatic stability of this locus was obtained from a set of experiments where we compared DNA-profiles from corresponding blood, semen and saliva samples. The results of this study suggest that in the near future analysis of the D1S80 locus by DNA-amplification can be applied in actual forensic case work.

RAPD (arbitrary primer) PCR is more sensitive than multilocus enzyme electrophoresis for distinguishing related bacterial strains

Abstract: The RAPD (random amplified polymorphic DNA) fingerprinting method, which utilizes low stringency PCR amplification with single primers of arbitrary sequence to generate strain-specific arrays of anonymous DNA fragments, was calibrated relative to the widely used, protein-based multilocus enzyme electrophoretic (MLEE) typing method. RAPD fingerprinting was carried out on five isolates from each of 15 major groups of Escherichia coli strains that cause diarrheal disease worldwide (75 isolates in all). Each group consisted of isolates that were not distinguishable from one another by MLEE typing using 20 diagnostic enzyme markers. In our RAPD tests, three or more distinct subgroups in each MLEE group were distinguished with each of five primers, and 74 of the 75 isolates were distinguished when data obtained with five primers were combined. Thus, RAPD typing is far more sensitive than MLEE typing for discriminating among related strains of a species. Despite their different sensitivities, the same general relationships among strains were inferred from MLEE and RAPD data. Thus, our results recommend use of the RAPD method for studies of bacterial population genetic structure and evolution, as well as for epidemiology.

Comparison of phage typing and DNA fingerprinting by polymerase chain reaction for discrimination of methicillin-resistant Staphylococcus aureus strains.

Abstract: A typing procedure for methicillin-resistant Staphylococcus aureus (MRSA) based on the polymerase chain reaction (PCR) amplification of both mecA sequences and variable DNA sequences as present in the prokaryotic genome has been developed. Two primers based on the sequences of DNA repeats as discovered in gram-negative members of the family Enterobacteriaceae allow detection of variable regions in the genome of a gram-positive bacterium such as S. aureus, as does a newly described arbitrary primer. This procedure, enabling the detection of 23 different genotypes in a collection of 48 MRSA isolates, was validated by comparisons with phage typing studies. It appeared that within the same group of isolates only 13 different phagovars could be identified. Combination of the results from both phage typing and genotyping allowed the discrimination of 34 of 48 isolates. However, depending on the primer-variable complexity of the PCR fingerprints, which could also be modulated by combination of PCR primers, clear homologies between the groups defined by either phage typing or fingerprinting were observed. An analysis of an MRSA outbreak in a geriatric institution showed a collection of genetically homogeneous isolates. In agreement with phage typing, PCR fingerprinting revealed the identical natures of the MRSA strains isolated from all patients. Images

Development of a rapid and efficient restriction endonuclease analysis typing system for Clostridium difficile and correlation with other typing systems.

Abstract: A HindIII restriction endonuclease analysis (REA) typing system for total genomic Clostridium difficile DNA including a rapid and efficient method of DNA extraction and a scheme for organizing unique electrophoretic DNA band patterns was developed. REA typing was performed by two extraction methods for 1,965 C. difficile isolates obtained from patients with symptomatic C. difficile disease, asymptomatic patients who were C. difficile culture positive, and environmental surfaces. This isolate collection yielded 206 unique REA types, which were organized into 75 groups. A reference strain representing each unique REA type was chosen for DNA band pattern comparisons, cytotoxin testing, and plasmid analysis. The DNA band patterns utilizing a guanidine thiocyanate-EDTA-Sarkosyl DNA extraction method were 94% reproducible, while the original and sporadically problematic diethyl pyrocarbonate-sodium dodecyl sulfate DNA extraction method was 98% reproducible when readable patterns were obtained. Reference strains from 43 of the 75 groups were cytotoxin positive, 28 groups were cytotoxin negative, and 4 groups included both toxigenic and nontoxigenic strains. Cytotoxicity of isolates with a particular REA type was always consistent with the toxicity of the reference strain for that type. REA typing was able to discriminate strain differences within types identified by the immunoblot (89 isolates), bacteriophage-bacteriocin (44 isolates), and ribotyping (23 isolates) methods. REA typing is a sensitive, discriminating, reproducible, and rapid method for differentiating C. difficile strains and is suitable for large-scale epidemiologic studies.

Typing of Staphylococcus aureus by pulsed-field gel electrophoresis, zymotyping, capsular typing, and phage typing: resolution of clonal relationships.

Abstract: Sixty-nine Staphylococcus aureus isolates from two epidemiologically unrelated sources were typed by pulsed-field gel electrophoresis after SmaI digestion of chromosomal DNA (genome typing), and the results were compared with those obtained by other typing methods: phage typing with the international set of phages, capsular serotyping with monoclonal antibodies against capsular polysaccharides type 5 and 8, and zymotyping by polyacrylamide agarose electrophoresis for esterase polymorphism. A good correlation of S. aureus types was found by these four typing methods. Differentiation increased in the order capsular typing < zymotyping < phage typing < genome typing, yielding 2, 10, 20, and 26 different S. aureus types, respectively. Five of the 26 genome types were further divided into several subtypes revealing clonal relationships. When 36 French S. aureus isolates were compared with 33 German S. aureus isolates, 3 strains representing clonal populations were identical in both groups. S. aureus isolates from patients with cystic fibrosis were also typed at the beginning and the end of a 4-week summer camp for these patients. The results suggested a possible strain transmission during the summer camp. We conclude that genome typing by pulsed-field gel electrophoresis is a powerful tool not only for strain identification but also for the resolution of the clonal relationships of S. aureus strains.

Comparison of PCR fingerprinting, by random amplification of polymorphic DNA, with other molecular typing methods for Candida albicans.

Abstract: SUMMARY: Fingerprinting by random amplification of polymorphic DNA (RAPD) was compared with existing molecular typing systems for Candida albicans. Fifteen isolates were chosen, including three from the same patient; these gave 14 distinct karyotypes by pulsed-field gel electrophoresis (PFGE) and 7 different DNA types by EcoRI-generated restriction fragment length polymorphisms (RFLPs). RAPD with primer I (5' GCT GGT GG3') gave 5 types, whereas primer II (5' GCG CAC GG3') yielded 11 types. Combining the results from both primers, all isolates were unique by RAPD with the exception of the three from the same patient. RAPD provided a fast, economical and reproducible means of typing C. albicans with a level of discrimination approaching that of PFGE.

Principal Typing Schemes in a Polyadic pi-Calculus

Abstract: The present paper introduces a typing system for a version of Milner's polyadic π-calculus, and a typing inference algorithm linear on the size of the input. The central concept underlying the typing system is the notion of type assignment, where each free name in a term is assigned a type, the term itself being given multiple nametype pairs. This observation leads to a clean typing system for Milner's sorting, and induces an efficient algorithm to infer the typing of a term. The typing system enjoys a subject-reduction property and possesses a notion of principal typing scheme. The algorithm to reconstruct the principal typing scheme of a process, or to detect its inexistence, is proved correct with respect to the typing system.

A review of the correlation of T-agglutination patterns and M-protein typing and opacity factor production in the identification of group A streptococci

Abstract: The classical techniques of M protein and opacity factor (OF) typing and T agglutination typing remain the "gold standard" in identifying group A streptococci, although newer techniques have been proposed to assist laboratory scientists, microbiologists, epidemiologists and clinicians in the precise identification and characterisation of these organisms. Because of the current scarcity of M-typing sera and the increased use by many laboratories of T typing as the sole method of group A identification, a table is presented to indicate specific correlation between the T-agglutination pattern and the M serotype. The use of this table will enable not only more selective use of typing sera but also, perhaps, result in improved understanding and ultimately in correlating these defined patterns with newer and more sensitive techniques.

Typing of Legionella pneumophila strains by polymerase chain reaction-mediated DNA fingerprinting.

Abstract: Well-defined Legionella pneumophila strains were analyzed by amplification of variable genomic regions with arbitrary and repeat sequence primers. Clinical and environmental outbreak-related isolates showed closely related amplicon patterns. Eleven strains of unrelated origins displayed 10 distinct patterns. Fingerprinting of L. pneumophila by polymerase chain reaction appeared to have the potential of being as epidemiologically useful as other genotypic methods. Images

Rapid, simple method for typing isolates of Mycobacterium tuberculosis by using the polymerase chain reaction.

Abstract: To develop a molecular typing method for Mycobacterium tuberculosis based on the polymerase chain reaction, oligonucleotide primers were designed to the ends of the insertion sequence IS6110 in an attempt to amplify DNA between clusters of this element on the genome. Although in many strains the copy number of this element is low and is distributed throughout the genome, most strains examined produced a banding pattern which varied between isolates including strains with one copy of IS6110. With strains isolated from patients in epidemiologic clusters of tuberculosis, the banding patterns were similar within each cluster but distinct from those in strains from different clusters. Similarly, multiple isolates from the same patient yielded a consistent banding pattern. Sequencing of four polymerase chain reaction products revealed that amplification was occurring between copies of IS6110 in two of the products and from a single copy of IS6110 to a nonspecific priming site in the other two. This technique provides a rapid and simple means of typing M. tuberculosis isolates for epidemiologic studies.

Detection, typing, and subtyping of enteric adenoviruses 40 and 41 from fecal samples and observation of changing incidences of infections with these types and subtypes.

Abstract: Monoclonal antibody (MAb) preparations specific for the enteric adenoviruses of subgenus F (AdF) were generated and evaluated as typing reagents in virus neutralization tests and enzyme-linked immunosorbent assays (ELISAs). A panel of 11 genome types of adenovirus 40 (Ad40), 24 genome types of Ad41, and 47 adenovirus prototype strains was used to determine the specificities of the MAbs in the two assays. In this way two MAbs, MAb 40-1 (anti-Ad40) and MAb 41-1 (anti-Ad41) were selected. These two MAbs showed strict type specificity in both assays. A third MAb reacted in an ELISA with all 47 human adenovirus types. With two other MAbs, three antigenic subtypes of Ad41 could be distinguished by their reactivities in virus neutralization tests and ELISAs. On the basis of the five selected MAbs, a sensitive ELISA system was developed for the direct detection and simultaneous typing and subtyping of Ad40 and Ad41 present in stool specimens. The five MAbs were also used to study the epidemiology of infections with Ad40 and Ad41 in The Netherlands in the period 1981 through 1989. It was shown that there were no significant fluctuations in the annual incidence of the cluster of enteric adenoviruses as a whole. This cluster should therefore be considered to belong to the "endemic" rather than the "epidemic" adenoviruses. The relative incidence of Ad40 infections compared with that of Ad41 infections changed considerably during the period studied; the proportion of Ad41 infections rose from about 30% in 1981 to about 95% in 1986, after which it stabilized at 90 to 95%. The proportion of one of the subtypes of Ad41 (Ad41 subtype M3) increased from about 40 to 80% in the same period.

Bacterial typing methods suitable for epidemiological analysis. Applications in investigations of salmonellosis among livestock.

Abstract: The ability to subtype bacteria by typing methods provides the bacteriologist with a powerful means to identify relationships between bacteria. This knowledge is used to identify routes of disease transmission among livestock and from livestock to humans. In the present paper, the principles of bacterial typing and the most commonly applied typing methods for use in veterinary public health are discussed in the context of their application in the investigation of salmonella epidemiology. Typing methods are now routinely used in most investigations on this subject and have provided insight into routes of transmission, reservoirs of infection and mechanisms of persistent infection. Under the EC order on zoonotic diseases, extended surveillance on the presence of zoonotic bacteria in livestock must be expected. To receive the maximum benefits of this surveillance, selected typing methods must be applied to all isolates of e.g. salmonella. At present, serotyping, and phage typing where applicable, are the most obvious choices for continuous surveillance of this organism. Random amplification of polymorphic DNA (PCR based typing) may have the potential for allocating strains into relevant groups quicker and without the requirement for additional manpower, and this method may be preferred in future.

Isoelectric focusing subtypes of HLA-A can be defined by oligonucleotide typing.

Abstract: This study describes a simple and direct method for sequence-specific oligonucleotide probe (SSOP) typing of the A locus of HLA class I genes. Genomic DNA from a panel of over 200 cells which have been characterized by the methods of serology and isoelectric focusing (IEF) for the HLA class I antigens was used for locus-specific PCR amplification of HLA-A sequences. Dot blot hybridization of the amplified products was performed with 28 SSOPs derived from hypervariable regions in exon 2 and 3. Co-amplification of three alleles of HLA-H pseudogene in apparent linkage disequilibrium with HLA-A2 and A10 was observed but did not interfere with the typing of HLA-A alleles. Using short SSOPs (15 nucleotides each) in single temperature tetramethylammonium chloride (TMAC) hybridization and washing steps, 30 IEF-definable isotypes of HLA-A antigens could be unambiguously defined by their hybridization patterns. Moreover, comparison of the typing results with available nucleotide sequences of HLA-A alleles showed that the conditions used allowed faithful detection of single codon mismatches between probe and template. Thus, these alleles can be identified by their unique hybridization patterns generated by the SSOPs. Nucleotide sequence analysis of any new HLA-A allele will further permit its rapid and unambiguous characterization by SSOP typing.

DNA typing of HLA-B gene in Takayasu's arteritis.

Abstract: Sixty-four patients with Takayasu's arteritis and 156 healthy individuals in the Japanese population were examined for HLA-B specificity at the DNA level by DNA typing using polymerase chain reaction (PCR)/sequence-specific oligonucleotide probe (SSOP) analysis and by subsequent sequencing analysis. The frequency of epitope combination group-B52 (EC-B52) corresponding precisely to HLA-B52 specificity and that of EC-B39.2 which is a newly-identified subtype of HLA-B39 specificity were increased in the patient group. These two disease-associated HLA-B alleles share an epitope composed of 63Glu and 67Ser. Because two HLA-B alleles, HLA-B51 and B39.1, which are similar but different at the epitope from HLA-B52 and B39.2, respectively, are not associated with Takayasu arteritis, 63Glu and 67Ser are supposed to be involved in the pathogenesis.

Molecular Methods for Microbial Identification and Typing

Abstract: General introduction. Analysis of nucleic acid profiles. DNA hybridization techniques. Nucleic acid amplification techniques. Polypeptide and LPS analysis. Application of antibodies to identification and typing. Electrophoretic multilocus enzyme analysis. Summary and future prospects. References. Index.

Defining the common subtypes of HLA A9, A10, A28 and A19 by use of ARMS/PCR.

Abstract: We describe sequence-specific primer (SSP) combinations for use in a one-step polymerase chain reaction (PCR) typing system to determine HLA-A locus subtypes of A9 (A23, A24), A10 (A25, A26, A43), A28 (A*6801, A*6802, A*6901) and A19 (A*2901, A*2902, A*3001, A*3002, A31, A32, A33) from genomic DNA. SSP's were designed on the basis of the amplification refractory mutation system (ARMS) in which a mismatch at the 3′ residue inhibits non-specific amplification. The SSP combinations described extend our low-resolution typing system, to provide a high-definition typing of the HLA-A locus.

Rapid, amplification-based fingerprinting of Mycobacterium tuberculosis

Abstract: Summary: Insertion element IS6110 occurs in multiple copies throughout the Mycobacterium tuberculosis genome, and the variability of its insertion sites is the basis for the IS6110 restriction fragment length polymorphism (RFLP) method for typing. We describe a novel gene amplification method to assess the variability of the location of IS6110. A unilateral-nested polymerase chain reaction and hybridization procedure was used to measure the variability in the distances between IS6110 elements and copies of a major polymorphic tandem repeat sequence of M. tuberculosis. The pattern of amplicons produced could be used to cluster epidemiologically related strains of M. tuberculosis into groups which correlated with the groups formed using IS6110-RFLP typing. Reliable patterns can be generated directly from sputum specimens as well as from M. tuberculosis cultures. We designated the novel method as IS6110-ampliprinting.

Comparison of Pulsed-Field Gel Electrophoresis with Isoenzyme Profiles as a Typing System for Candida tropicalis

Abstract: Candida species are important nosocomial pathogens, particularly in immunocompromised and critically ill patients. A variety of methods have been used to differentiate strains, but an optimal system has not been established. We compared methods for typing a panel of nine related isolates of Candida tropicalis from an outbreak of sternal wound infections as well as four unrelated control isolates of this species. (The genetic relationships of the nine isolates in the panel had been confirmed previously by restriction fragment analysis.) Typing was undertaken without knowledge of an isolate's origin. Karyotyping by contour-clamped homogeneous electric field (CHEF) gel electrophoresis failed to distinguish between outbreak and control isolates. However, when chromosome-sized DNA was digested with SfiI, EagI, SacII, or NaeI and the fragments were separated by CHEF electrophoresis, the outbreak isolates were readily identified. The isoenzyme profiles of the outbreak isolates were identical and were distinctly different from those of the control isolates. While both isoenzyme profiles and the modified CHEF procedure were discriminatory, the latter is recommended as a relatively convenient and reproducible technique for comparison of types of C. tropicalis.

Typing of human rotavirus VP4 by an enzyme immunoassay using monoclonal antibodies.

Abstract: Two different neutralization specificities exist on the outer capsid of group A rotaviruses. At least seven VP7 (G) antigenic types are distinguishable among human rotaviruses. Four distinct antigenic (P) types of human rotavirus VP4 corresponding to separate rotavirus gene 4 groups have been described. The aim of this study was to identify P types in clinical specimens by developing an enzyme immunoassay, using P-type-specific neutralizing monoclonal antibodies (N-MAbs). Three N-MAbs primarily or solely recognizing each of P types 4, 6, and 8 and binding to VP4 or its subunit VP5* were derived. These N-MAbs served as detector antibodies in an enzyme immunoassay P-typing system similar to that in use for G typing. P-type specificity was highest when the G-type specificity of the capture antiserum was matched to the G type of the rotavirus in the test sample. The method correctly identified the P types of 13 well-characterized, cell culture-adapted human rotaviruses and was used to classify a further six strains. P typing of 118 rotavirus-positive stools gave results consistent with the P type inferred from the G type for 98 (83%) samples. Twelve (10%) of the stools showed no reaction with any N-MAb and eight (7%) samples were untypeable because of cross-reactivity between N-MAbs or high background readings. This P-typing enzyme immunoassay system is economical and amenable to large-scale use in epidemiological studies. Its use will facilitate assessment of the distribution of P types worldwide and of the role of VP4 in eliciting protective immune responses.

Genetic analysis using polymerase chain reaction-amplified DNA and immobilized oligonucleotide probes: reverse dot-blot typing.

Abstract: The reverse dot-blot method is a simple and rapid diagnostic procedure that allows screening of sample for a variety of mutations/polymorphisms in a single hybridization reaction. Several methods of immobilizing the oligonucleotide probes are discussed. The reverse dot-blot method has several unique properties that are valuable in a diagnostic setting: (1) the typing results from a single sample can be located on a single strip. This facilitates scanning and interpretation of the probe reactivity patterns and minimizes the potential for user error. (2) The test can utilize premade typing strips. This minimizes user labor as well as error potential and allows the use of standardized reagents. (3) Unlike dot-blot/oligonucleotide typing, only the PCR product is labeled, eliminating the potential problem of probes labeled to different specific activities. This method has already been used in the areas of forensic genetic typing (the HLA-DQ alpha Amplitype test), tissue typing for transplantation (the HLA-DR beta) test, cystic fibrosis screening, as well as in a variety of research applications.