Showing papers on "Typing published in 1991"

Typing of deoxyribonucleic acid (DNA) extracted from compact bone from human remains.

Abstract: The application of deoxyribonucleic acid (DNA) typing methods for the potential identification of unknown human remains was investigated. DNA was isolated from compact bone tissue from badly decomposed bodies and from known and unknown human remains, using a decalcification and ion wash procedure. Restriction fragment length polymorphism (RFLP) analysis of variable number of tandem repeats (VNTR) loci yielded results in some cases, but more often the DNA was too degraded to produce RFLP patterns. No RFLP profiles could be obtained from putrified soft tissues. However, DNA extracted from compact bone tissue of human remains up to eleven years old was successfully amplified using the polymerase chain reaction (PCR) for the VNTR loci D1S80, D17S5, COL2A1, and APO B, as well as the HLA-DQ alpha locus. This is especially significant, since PCR results were obtained from those samples whose DNA had been degraded substantially and had yielded no RFLP patterns. All DNA types determined from the compact bone tissue from decomposed bodies whose identification had been established first by other means (and whose parents or offspring were available for typing) demonstrated mendelian inheritance of the alleles of the loci analyzed. These results suggest that amplification and typing of DNA extracted from compact bone of human remains could be useful in establishing the identity of a person, as well as in excluding possible false identifications.

HLA-DR, DQ and DP typing using PCR amplification and immobilized probes.

Abstract: A simple, rapid, and precise method of typing HLA class II polymorphism would be valuable in the areas of disease susceptibility, tissue transplantation, individual identification and anthropological genetics. Here we describe a method of analysing class II sequence polymorphism based on polymerase chain reaction (PCR) amplification and hybridization with oligonucleotide probes. One valuable property of sequence-based HLA typing strategies, like oligonucleotide probe hybridization, is that they reveal how and where two alleles differ, not simply that they can be operationally distinguished. The nature and location of HLA polymorphisms appears to be critical in disease association studies and are likely to be important in tissue typing for transplantation. New alleles at the DRB1, DPB1 and DQB1 loci are likely to be identified as this technology is applied to more and more samples, particularly in non-Caucasian ethnic groups. A new allele is uncovered as an unusual pattern of probe binding and then confirmed by sequencing. This pattern is observed because class II polymorphism is localized to specific regions and virtually all 'new' alleles have polymorphisms in the region of probe binding. Obviously, any new allele with a new polymorphic sequence in a region for which typing probes are not available would not be revealed by oligonucleotide typing. With the PCR primers and probes described here, 7 DQA1 alleles, 15 DQB1 alleles, 18 DPB1 alleles, and 32 DRB1 alleles are distinguished. Additional primers and/or probes can, of course, increase the allelic discrimination of oligonucleotide dot blot typing. These horseradish peroxidase (HRP)-labelled oligonucleotide probes are stable (greater than 2 years when stored at 4 degrees C) and the typing system is simple and robust. Over 500 samples from the CEPH pedigrees (unpublished data; A. B. Begovich, et al., manuscript in preparation) and greater than 1000 unrelated samples have been typed by this procedure. Although this dot blot/oligonucleotide hybridization procedure is a powerful and precise method of HLA class II typing, the complexity of the procedure increases as the number of probes required for analysis increases. The reverse dot blot method, based on an array of immobilized probes, allows the typing of individual samples in one single hybridization reaction. In this approach, a panel of unlabelled oligonucleotides are immobilized to a nylon membrane. The PCR product is labelled during the amplification reaction by using biotinylated primers and hybridized to the membrane. The presence of bound PCR product specifically hybridized to a given probe is detected using streptavidin-HRP conjugates and either chromogenic or chemiluminescent substrates.(ABSTRACT TRUNCATED AT 400 WORDS)

Phage typing of Salmonella enteritidis in the United States.

Abstract: The number of reported isolates of Salmonella enteritidis has increased dramatically in the last 10 years. For many years phage typing has been a useful epidemiologic tool for studying outbreaks of S. typhi and S. typhimurium. In 1987, Ward et al. (L. R. Ward, J. De Sa, and B. Rowe, Epidemiol. Infect. 99:291-294, 1987) described a phage typing scheme for S. enteritidis. This system differentiated 27 phage types by use of 10 typing phages. With these phages, we typed 573 strains of S. enteritidis from humans (42 outbreaks), animals, food, and the environment. Ninety-six percent of the strains were typeable. The most common phage types were 8 (48.2%), 13a (20.1%), 13 (7.8%), and 14b (7.8%). Most of the strains were specifically collected from egg-related outbreaks in the northeastern United States in 1988 and 1989, probably accounting for the distribution of the four most common types in this sample. This system was particularly useful for differentiating a group of animal strains that had a number of diverse phage types. For 49 animal strains typed, 16 different patterns were obtained. Phage type 8 represented 32% of these strains, but no other phage type represented more than 8% of these strains. One-half of the 16 animal strains that were phage type 8 were from poultry. This phage typing system will be useful for comparing phage types found in the United States with those types encountered worldwide and for determining whether virulent strains of phage type 4 are entering the United States. Additional phage typing systems as well as molecular techniques are being studied to determine whether they can differentiate strains of phage types 8 and 13a.

PCR-based typing of DNA extracted from cigarette butts

Abstract: Limited genetic marker information can be obtained from saliva by typing by conventional serological means. Thus, the application of PCR-based DNA typing methods was investigated as a potential approach for typing genetic markers in saliva. DNA was isolated from 200 cigarettes smoked by 10 different individuals (20 cigarettes per individual) and from 3 cigarette butts recovered from 2 crime scenes (adjudicated cases) using a Chelex 100 extraction procedure. The amount of recovered human DNA was quantified by slot-blot analysis and ranged from approximately less than 2-160 ng DNA per cigarette butt for the 200 samples, and 8 ng, 50 ng, and 100 ng for the cigarette butts from the adjudicated cases. The DNA was successfully amplified by the polymerase chain reaction (PCR) for the HLA-DQ alpha locus (99 out of 100 samples) as well as for the variable number of tandem repeat (VNTR) locus D1S80 (99 out of 100 samples). Amplification and typing of DNA was successful on all samples recovered from the crime scenes. The results suggest that PCR-based typing of DNA offers a potential method for genetically characterizing traces of saliva on cigarette butts.

HLA-DRB1*01 subtyping by allele-specific PCR amplification: a sensitive, specific and rapid technique.

Abstract: The two DR1-associated cellular specificities Dw1 and Dw20, as well as DR'Br' (Dw'BON'), cannot be unequivocally assigned by serological typing or restriction fragment length polymorphism (RFLP) analysis. We have developed and compared two polymerase chain reaction-based (PCR) typing methods for distinguishing these DRB1 alleles; allele-specific amplification of DRB1*01 alleles followed by an agarose gel electrophoresis detection step and group-specific DRB1*01 amplification followed by hybridization with sequence-specific oligonucleotide probes. The two typing strategies gave completely concordant results in the 33 DRB1*01-positive and the 46 DRB1*01-negative individuals and cell lines studied. No false-negative or false-positive typing results were obtained. All possible heterozygous combinations of the DRB1*0101-0103 alleles could be distinguished by both typing methods. DRB1*01 subtyping by allele-specific PCR amplification was performed in less than 3 hours, including PCR amplification, detection and interpretation steps. The technique will be a valuable complement to DR typing by serology and RFLP analysis. Allele-specific DRB1 amplifications or group-specific amplifications followed by directed allele-specific amplifications of DRB1 alleles, typing based on the absence or presence of amplified products, may well prove to be the technical innovation that will firmly establish PCR-based DR typing in routine clinical tissue typing.

Validation Studies on the Analysis of the HLA DQα Locus Using the Polymerase Chain Reaction

Abstract: A series of experiments has been performed to evaluate typing of the HLA DQ alpha gene by polymerase chain reaction (PCR) amplification of the gene and subsequent hybridization with sequence-specific oligonucleotide probes. These experiments were designed to evaluate DQ alpha typing for analysis of evidentiary specimens. Bloodstains were exposed to a variety of conditions and environmental insults. These conditions included exposure to many different types of substrates, various microorganisms that could be encountered in evidentiary stains, sunlight, and a variety of chemical contaminants. Varying amounts of genomic deoxyribonucleic acid (DNA) were amplified to test the sensitivity of DQ alpha typing. The sensitivity of the PCR technique raises the concern that DNA from sources other than the evidentiary material could be detected. A series of experiments was done to evaluate the question of DNA contamination. Purified DNA samples with different DQ alpha types were mixed in different ratios to determine the ratio at which it could not be determined whether an allele was from the sample or the contaminant. Samples were exposed to a variety of situations that could lead to contamination, such as extensive handling and exposure to coughing or sweaty clothing, to other wet bloodstains, and to saliva. The DQ alpha types were determined from 469 individuals from three sample populations (Caucasian, black, and Hispanic), and the genotype frequencies were compared with frequencies previously reported by others. DNA samples from old cases [which had previously been analyzed by restriction fragment length polymorphism (RFLP) typing of variable number of tandem repeat sequences] were typed. All samples that were excluded by DQ alpha typing were also excluded by RFLP analysis, and all samples that were included by RFLP analysis were included by DQ alpha typing. Finally, the problem of allele dropout, or the failure to detect particular alleles, was noted and alleviated by performing the typing under appropriate conditions. The results of these validation experiments indicate that typing of the DQ alpha gene by PCR and detection of specific alleles can be accomplished, when the typing is done using proper protocols, without producing false positive or false negative results.

A critical review of typing methods for Candida albicans and their applications.

Abstract: During the 1980s, a large number of typing methods for the strain differentiation of Candida albicans were described in the literature. Although these methods have been based on a variety of physiological and genetic markers, none is ideal. This review discusses the characteristics of an ideal typing method in terms of its typability, reproducibility, and discriminatory power. Ways of determining these characteristics are presented so that the available typing methods for Candida albicans can be objectively compared. Available typing methods for C. albicans include serotyping, morphotyping, resis-totyping, biotyping, and killer yeast typing. Electrophoretic methods include immunoblotting, isoenzyme analysis, analysis of DNA restriction fragment length polymorphism, karyotyping, and the use of DNA probes. The application of these methods to epidemiological research, the investigation of outbreaks of disease, and die study of virulence is described. The potential impact of the phenomenon of phenotyp...

Comparison of restriction enzyme analysis and pulsed-field gradient gel electrophoresis as typing systems for Candida albicans.

Abstract: Candida species are an important cause of infection in immunocompromised hosts and the leading cause of nosocomial fungal infections. Study of the epidemiology of Candida infection has been difficult because of lack of a reliable typing system. We describe a typing system utilizing contour-clamped homogeneous electric fields (CHEF), which is a modified version of pulsed-field gradient gel electrophoresis, and compared it with restriction enzyme analysis (REA) of genomic DNA. The study was done with 35 Candida albicans clinical isolates from separate patients. CHEF and REA were performed on each isolate, and the patterns were compared. The REA procedure revealed 17 strain types while the CHEF procedure was able to distinguish 23 strain types of C. albicans. The CHEF technique yields unique patterns of chromosomal bands that can be used to distinguish clinical isolates and demonstrates greater sensitivity than REA. Images

Improved procedure for bacteriophage typing of Listeria strains and evaluation of new phages

Abstract: A modified technique in listerial phage typing, termed reversed phage typing procedure, was developed Ready-to-use typing plates were prepared by preapplication of phage suspensions on tryptose agar plates The new procedure offers a number of substantial advantages as compared with the conventional method, being much more reliable, efficient, and convenient to use More than 1,000 strains of Listeria have thus far been typed with the reversed phage typing procedure, employing an extended set of 21 genus-specific bacteriophages The overall typability of strains was 895%

Typing of chlamydia trachomatis by restriction endonuclease analysis of the amplified major outer membrane protein gene

Abstract: A procedure was developed for characterization of Chlamydia trachomatis strains by using restriction endonuclease analysis of amplified genes of the major outer membrane protein (MOMP) Reference strains of the 15 serovars (A through K and L1 through L3) and clinical isolates were tested The nucleotide sequences of the MOMP genes of each of the 15 serovars were arbitrarily constructed by using the sequences of the four variable domains known for each serovar and the constant domains of serovar L1 Computer analysis of these sequences indicated that two restriction digestions performed in parallel, one with AluI and the other with IIpaII, followed by HinfI and EcoRI, would allow the theoretical differentiation of 13 serovars Serovars Ba and L1 presented the same theoretical restriction profile Our typing method consisted of polymerase chain reaction amplification of a fragment of about 1,200 bp of the MOMP gene, followed by restriction endonuclease digestion with the aforementioned enzymes From the 15 serovars, we obtained 14 different patterns; 13 profiles were serovar specific, while serovars B and Ba presented the same pattern Application of this typing method to C trachomatis strains isolated from clinical material gave the same results as the immunotyping method for 14 of 17 strains Furthermore, restriction endonuclease analysis detected differences within a serovar This method seems to be promising for epidemiological studies

Epidemiologic and clinical utility of typing systems for differentiating among strains of methicillin-resistant Staphylococcus aureus.

Abstract: Typing systems for differentiating among strains of methicillin-resistant Staphylococcus aureus (MRSA) can be valuable tools for the epidemiologist and the clinician. Specific criteria for evaluating such systems are typeability, reproducibility, and discriminatory power. An ideal typing system also would be rapid, inexpensive, technically simple, and readily available. Systems based on the detection of phenotypic variations include antimicrobial susceptibility testing, bacteriophage typing, multilocus enzyme electrophoresis, and electrophoretic methods such as protein eletrophoresis and immunoblotting. Systems that directly detect genotypic variations include plasmid profile analysis, restriction enzyme analysis of plasmid DNA, restriction enzyme analysis of chromosomal DNA, Southern blot analysis of specific restriction fragment length polymorphisms, and pulse field gel electrophoresis. in general, the more widely available typing systems based on phenotypic assays and plasmid analysis have limitations in typeability and/or discriminatory power.The chromosomal DNA-based techniques, although promising, are unproven approaches still under active investigation.

Comparison of ribotyping with conventional methods for the type identification of Enterobacter cloacae.

Abstract: An Escherichia coli rRNA probe was compared with a combination of O serotyping, phage susceptibility, and biotype pattern for the type identification of strains of Enterobacter cloacae Forty-five isolates of E cloacae from 36 patients in nine hospitals were examined By conventional typing, only 26 (577%) could be assigned to a specific serotype and 6 (133%) were autoagglutinating owing to rough lipopolysaccharide antigens All isolates could be assigned to one of three biotypes, but many phage sensitivity patterns were evident Twenty-nine distinct strains were identified by combined typing Probing of EcoRI and BamHI digests of chromosomal DNA with a cDNA copy of E coli rRNA proved to be highly discriminating between strains Thirty different ribotypes based on 28 bands were recorded Overall, agreement between the ribotyping and combined typing methods was good (844%), and discrepancies were generally confined to serologically unclassifiable strains and variability in biotype codes Ribotyping was reproducible, and five of six pairs of isolates from the same and different patients gave identical hybridization profiles on separate occasions We conclude that ribotyping is a highly discriminatory and reproducible method for the typing of E cloacae, but in most outbreaks it offers little increase in discrimination over traditional methods

A new typing method for the avian infectious bronchitis virus using polymerase chain reaction and restriction enzyme fragment length polymorphism.

Abstract: Two primers with the length of 22 bases each and 400 bases apart on the spike protein gene of avian infectious bronchitis virus (IBV) were prepared. Using these primers, the genome RNA from twelve strains of the various serotypes were reverse-transcribed to cDNA and amplified by polymerase chain reaction (PCR). With all strains, 400 base DNA was amplified, indicating that there were no apparent insertions or deletions in this region. However, the amplified DNA showed different cleavage patterns by the restriction enzymes. These 12 strains were classified into 5 groups. The strain typing based on a comparison of the cleavage patterns was consistent with the previous serological typing. This study thus provides a simple and rapid method for typing of IBV.

Genome fingerprinting as a typing method used on polyagglutinable Pseudomonas aeruginosa isolates from cystic fibrosis patients.

Abstract: Phenotypical changes occur in the surface of Pseudomonas aeruginosa during the chronic lung infection of cystic fibrosis patients. It is difficult with the classical typing methods, such as serotyping, phage typing and pyocin typing, to decide if a patient has been colonized with a new strain or whether it is the same strain which has reappeared, for instance after chemotherapy in the lungs. This investigation was carried out to evaluate genome fingerprinting as a typing method and to see how it correlated with classical methods and with DNA probe typing. Forty Pseudomonas aeruginosa isolates, 34 polyagglutinable and six monoagglutinable, from 14 cystic fibrosis patients were analysed using genome fingerprinting. The bacterial chromosomes were digested with the restriction endonucleases Dra 1 and Xbal, and separated by field inversion gel electrophoresis. The results were compared with those of a previous work (Ojeniyi et al. 1990) concerning typing with a DNA probe, serotyping using both polyclonal and monoclonal sera, phage typing, pyocin typing and reverse phage typing. The results of genome fingerprinting and DNA probe typing showed the best correlation, followed by pyocin typing. The correlation between the results of genome typing and the other typing methods was low. The discriminatory effect of genome fingerprinting was higher than that of DNA probe typing, and genome fingerprinting was found to be the best single method for epidemiological investigations of polyagglutinable isolates from cystic fibrosis patients.

Non-radioactive restriction fragment length polymorphism (RFLP) typing of Clostridium difficile

Abstract: A typing method for Clostridium difficile based on restriction fragment length polymorphisms (RFLP) is described. The technique utilizes commercially available Escherichia coli ribosomal ribonucleic acid (rRNA) as probe material. Probe labelling, hybridization and detection was performed using the Enhanced Chemiluminescence (ECL) gene detection system. The probe labelling procedure was easy to perform, taking only 20 min. The complete typing method was comparatively simple, reproducible and readily adaptable to most bacterial genera.

Methods and reagents for hla drbeta dna typing

Abstract: Primers for amplification of specific nucleic acid sequences of the second exon of HLA DRbeta genes and probes for identifying polymorphic sequences contained in the amplified DNA can be used in processes for typing homozygous or heterozygous samples from a variety of sources and for detecting allelic variants not distinguishable by serological methods. This HLA DRbeta DNA typing system can be used in a dotblot format that is simple and rapid to perform, produces detectable signals in minutes, and can be used for tissue typing, determining individual identity, and identifying disease susceptible individuals.

Positive correlation between oligonucleotide typing and T-cell recognition of HLA-DP molecules.

Abstract: The identification of 19 different HLA-DPB1 sequences implicates the existence of more DP specificities than can be typed for with cellular methods. How many of the DP beta sequences can be specifically recognized by T cells, and which of the polymorphic regions can contribute to the specificity of allorecognition, is not known. In order to investigate the distribution and the immunological relevance of recently described DPB1 alleles, we have typed a panel of 98 randomly selected Dutch Caucasoid donors for the HLA-DPB1 locus by oligonucleotide typing. Comparison of the typing results with primed lymphocyte typing (PLT) defined DP specificities shows an extremely good correlation. Moreover, additional alleles could be defined by oligonucleotide typing reducing the number of DP blanks in the panel. By selecting the appropriate responder stimulator combinations we were able to show that distinctive PLT reagents against oligonucleotide defined specificities DPB1*0401, DPB1*0402, DPB1*0901, and DPB1*1301 can be generated. To investigate in more detail which part of the DP molecule is responsible for the specificity of T-cell recognition, T-cell clones were generated against HLA-DPw3. The clones were tested for the recognition of stimulators carrying DPB1 alleles which had been defined by oligonucleotide typing and sequence analyses and which differed in a variable degree from DPB1*0301. The recognition patterns demonstrated that differences of one amino acid in polymorphic regions situated either in the beta sheets or alpha helix of the hypothetical model of the HLA class II molecule can eliminate T-cell recognition. Furthermore, sequence analyses revealed a new DPB1 allele designated DPB1*Oos.

Heterogeneity, persistence, and distribution of Pseudomonas aeruginosa genotypes in cystic fibrosis patients.

Abstract: A collection of 222 isolates of Pseudomonas aeruginosa was obtained from the respiratory tract of 16 patients with cystic fibrosis over a 4- to 9-month period. Fourteen of these patients were unrelated, while the remaining two were siblings. Isolates were typed by conventional pyocin typing and also by the use of a DNA probe containing 741 bp immediately upstream of the exotoxin A structural gene and the initial 732 bp of the exotoxin A structural gene. By pyocin typing, 69% (11 of 16) of the patients were shown to harbor a single type that persisted in the lung throughout the study. By genotyping (DNA probe typing), all but three patients (13 of 16, 81%) harbored a single persistent genotype in their lungs. Six patients other than the sibling pair (6 of 14, 43%) shared a common genotype in their lungs as judged by DNA probing, and the pyocin type of these isolates was also identical. In four of these six patients, the shared genotype was also the persistent genotype. The sibling pair studied also carried a common genotype in their lungs as indicated by DNA probing, even though the pyocin type of these isolates varied. Results presented suggest that the majority of patients harbor a persistent strain in their lungs and that cross-colonization may occur.