Showing papers on "Typing published in 1992"

HLA-DR typing by PCR amplification with sequence-specific primers (PCR-SSP) in 2 hours: an alternative to serological DR typing in clinical practice including donor-recipient matching in cadaveric transplantation.

Abstract: In most PCR-based tissue typing techniques the PCR amplification is followed by a post-amplification specificity step. In typing by PCR amplification with sequence-specific primers (PCR-SSP), typing specificity is part of the amplification step, which makes the technique almost as fast as serological tissue typing. In the present study primers were designed for DR "low-resolution" typing by PCR-SSP, i.e. identifying polymorphism corresponding to the serologically defined series DR1-DRw18. This resolution was achieved by performing 19 PCR reactions per individual, 17 for assigning DR1-DRw18 and 2 for the DRw52 and DRw53 superspecificities. Thirty cell lines and 121 individuals were typed by the DR "low-resolution" PCR-SSP technique, TaqI DRB-DQA-DQB RFLP analysis and serology. The concordance between PCR-SSP typing and RFLP analysis was 100%. The reproducibility was 100% in 40 samples typed on two separate occasions. No false-positive or false-negative typing results were obtained. All homozygous and heterozygous combinations of DR1-DRw18 could be distinguished. Amplification patterns segregated according to dominant Mendelian inheritance. DNA preparation, PCR amplification and post-amplification processing, including gel detection, documentation and interpretation, were performed in 2 hours. In conclusion, PCR-SSP is an accurate typing technique with high sensitivity, specificity and reproducibility. The method is rapid and inexpensive. DR "low-resolution" typing by the PCR-SSP technique is ideally suited for analyzing small numbers of samples simultaneously and is an alternative to serological DR typing in routine clinical practice including donor-recipient matching in cadaveric transplantations.

Identification of group A rotavirus gene 4 types by polymerase chain reaction.

Abstract: Five genetically distinct human rotavirus (HRV) gene 4 groups have been described on the basis of comparative nucleotide sequencing and the predicted amino acid sequences, and at least four of them represent distinct VP4 antigenic types. To identify each gene 4 type and investigate its distribution in HRV isolates from patients with diarrhea, we developed a polymerase chain reaction (PCR) typing method using sequence information available for four genetically distinct gene 4 types. Rotavirus double-stranded RNAs (dsRNAs) isolated from stool samples were first reverse transcribed and amplified by PCR by using two oligonucleotide primers that correspond to regions that are highly conserved among all known HRV gene 4 types. The 876-bp dsDNA products were then reamplified by PCR in the presence of a cocktail containing one conserved plus-sense primer and four type-specific minus-sense primers (selected from the hypervariable region of gene 4), resulting in products of 345, 483, 267, and 391 bp corresponding to gene 4 types 1, 2, 3, and 4, respectively. This method reliably identified the gene 4 types of 16 well-characterized HRV isolates. Our results were independently confirmed for all 16 strains by reverse transcription and PCR amplification of HRV dsRNA in the presence of alternate type-specific primer pairs. For direct gene 4 typing of HRV in stool samples, we developed a method to extract rotavirus dsRNA from stool specimens by using glass powder. Our results suggest that gene 4 typing will be useful in providing more a complete characterization of HRV strains of epidemiologic or vaccine-related interest.

Molecular typing of Staphylococcus aureus on the basis of coagulase gene polymorphisms.

Abstract: Staphylocoagulase, a major phenotypic determinant of Staphylococcus aureus, exists in multiple allelic forms, in part because of the existence of gene variants within the 3'-end coding region. This region contains a series of repeating 81-bp DNA sequences which differ both in the number of tandem repeats and the location of AluI restriction sites among different isolates. Utilizing this finding, we developed a novel typing method for S. aureus based on polymerase chain reaction amplification of the variable region of the coagulase gene followed by AluI restriction enzyme digestion and analysis of restriction fragment length polymorphism (RFLP). Among 30 S. aureus isolates studied initially, a total of 10 distinct RFLP patterns were observed. There was excellent correlation of the RFLP patterns with typing of these isolates by multilocus enzyme electrophoresis at 20 chromosomal loci. This coagulase RFLP method was used to analyze an additional 39 S. aureus isolates and successfully traced the source of an outbreak of methicillin-resistant S. aureus infections at a local hospital.

Polymerase Chain Reaction (PCR) Amplification and Human Leukocyte Antigen (HLA)-DQα Oligonucleotide Typing on Biological Evidence Samples: Casework Experience

Abstract: The polymerase chain reaction (PCR) method of specific gene amplification was used in casework to synthesize millions of copies of the polymorphic second exon of the human leukocyte antigen (HLA)-DQ alpha (or DQA1) locus from a variety of evidence samples The HLA-DQ alpha allelic variants in the amplified deoxyribonucleic acid (DNA) were determined in a rapid non-radioactive test by hybridization to sequence-specific oligonucleotide probes in both the dot-blot and reverse dot-blot formats This genetic typing system has been subjected to blind proficiency testing; the performance of this test in the analysis of experimentally mixed samples was also evaluated As of August 1990, over 250 cases have been tested and more than 2000 individual evidence (bloodstains, semen stains, individual hairs, bone fragments, and tissue sections) and reference samples have been analyzed The first 198 of these cases are summarized in this paper; in 65% of the cases with conclusive results a suspect was included, and in 35%, all suspects were excluded Individual cases as well as some of the general issues relating to forensic science analysis and this genetic typing system are discussed The high rate of exclusion reported here combined with the ability of PCR to type old evidence samples suggests the relevance of this genetic test for postconviction review; two cases in which the convicted suspect was excluded are discussed

Genotyping of Chlamydia trachomatis from a Trachoma-Endemic Village in The Gambia by a Nested Polymerase Chain Reaction: Identification of Strain Variants

Abstract: Direct amplification of the major outer membrane protein (MOMP) gene by polymerase chain reaction (PCR) was used to identify Chlamydia trachomatis in eye swabs from clinically active cases of endemic trachoma in a Gambian village. Chlamydial DNA was detected in 51% of 96 subjects with clinically active disease and in 5% of 37 clinically negative individuals. The PCR detection was combined with typing, using nested primers to variable sequences (VS) 1, 2, and 4 of the MOMP genes to distinguish between trachoma genotypes A, B, and C, respectively. Genotypes A and B were detected in the village, with some individuals harboring both genotypes within the same eye. DNA sequencing revealed strain variants of both genotypes. Typing of genotype and strain variants is now in progress to study trachoma transmission within the village.

Typing of Enterococcus species by DNA restriction fragment analysis.

Abstract: Enterococci are a frequent cause of hospital-acquired infection, being associated with urinary tract infections, wound sepsis, bacteremia, and endocarditis. The source of infection is usually thought to be endogenous, but some evidence points to cross-infection between patients. A better understanding of the epidemiology of enterococci has been limited by the lack of a good discriminatory typing system. This report describes the application of two DNA-based typing methods to Enterococcus faecalis and Enterococcus faecium: comparison of restriction fragments from total DNA by conventional electrophoresis and comparison of restriction fragments hybridizing to an rRNA gene probe (ribotyping). Comparison of restriction fragments (from SstI digestion) by conventional electrophoresis was simple and highly discriminatory. The results of analysis of blood culture isolates and of repeat isolates from individual patients are reported. Ribotyping (with BscI digestion) was more applicable at the level of species discrimination.

Biochemical and molecular characterization of Salmonella enterica serovar berta, and comparison of methods for typing.

Abstract: Strains of Salmonella enterica serovar berta (S. berta) from Denmark and seven other countries have been characterized with the aim of developing a rational typing strategy in connection with outbreak investigations. Biotyping divided the strains into H2S-positive (90%) and H2S-negative (10%) biovars. Six percent of the strains were resistant to one or more antimicrobial agents. Eighty-eight percent of the strains carried plasmids and 52 different plasmid profiles were recognized. Six of the common plasmid sizes in these profiles were shown by restriction enzyme analyses to contain more than one plasmid species. More than 90% of the strains had the same ribotype with the restriction enzymes Sma I and EcoR I and the same whole cell protein profile. Outer membrane protein profiles and isoenzyme profiles were identical in all S. berta analysed. Plasmid profiling in combination with restriction enzyme analysis of plasmids seemed to be the most rational typing strategy for S. berta. The results indicated that S. berta strains regardless of geographical source or host are possibly clonal in nature.

Ribotyping of coagulase-negative staphylococci with special emphasis on intraspecific typing of Staphylococcus epidermidis.

Abstract: Coagulase-negative staphylococci (CoNS), particularly Staphylococcus epidermidis, are increasingly being recognized as opportunistic pathogens. They are often multiply antibiotic resistant and can cause nosocomial outbreaks. For clinical and epidemiological reasons, accurate species identification and typing are imperative. Ribotyping, i.e., the generation of characteristic fragment patterns by hybridization of restriction endonuclease fragments of total DNA with labeled standard rRNA from Escherichia coli, has been applied to CoNS for species identification by various investigators. The present study, involving 115 randomly collected clinical isolates of CoNS, provides ambiguous evidence with respect to those findings. Eighty six S. epidermidis strains were ribotyped intraspecifically. Eleven different ribotypes were found after digestion with EcoRI, and 10 were found with HindIII. A combination of the two restriction endonucleases resulted in an increase in the discriminatory power (DP) from 14.3 to 31.6%. A combination of ribotyping with biotyping raised the DP to a maximum of 48.6%. The reproducibility of ribotyping was 100% after greater than 400 generations of growth. No correlation between methicillin resistance and certain ribotypes among the S. epidermidis strains was observed. Ribotyping is considered a useful tool for the intraspecific typing of CoNS for epidemiological purposes. The DP can be increased by the use of additional restriction endonucleases.

Typing of toxic strains of Clostridium difficile using DNA fingerprints generated with arbitrary polymerase chain reaction primers.

Abstract: Clostridium difficile is the causative agent for pseudomembranous colitis in humans. Toxic strains of C. difficile produce two toxins, toxin A and toxin B. A reliable and definitive method of typing the toxic strains of C. difficile is needed since nosocomial cross infection is a primary concern in hospitals and other health care facilities. A method for typing toxic strains of Clostridium difficile using arbitrary polymerase chain reaction (PCR) primers is presented in this study. The C. difficile strains were initially characterized for the toxin A genetic determinant using specific PCR primers which differentiate toxin positive from toxin negative strains. These toxic strains were then PCR typed using six arbitrary primers which generated DNA patterns that were unique for all toxic strains examined. The use of this typing scheme in clinical applications is discussed.

Identification of meningococcal serosubtypes by polymerase chain reaction.

Abstract: The polymerase chain reaction was used as the basis of a novel typing method for Neisseria meningitidis. Southern hybridization experiments demonstrated that it was possible to identify genes encoding different serological variants of the meningococcal class 1 outer membrane protein by probing with polymerase chain reaction products corresponding to known epitopes. A set of 14 defined variable regions was prepared in bacteriophage M13mp19 by the cloning of polymerase chain reaction products. The phage were dot blotted onto membrane filters, which were used as targets for hybridization of radiolabeled amplified class 1 outer membrane protein genes. Thus, the presence of many different subtype-specific epitopes could be investigated in one experiment. This technique was evaluated with a set of serological reference strains, mainly of serogroup B organisms, and provided an alternative, rapid, and comprehensive typing system that was capable of distinguishing known serosubtypes and also of defining currently untypeable strains independently of sodium dodecyl sulfate-polyacrylamide gel electrophoresis or serological analysis. An additional advantage of this technique was that in the case of an unknown serosubtype (i.e., one that did not hybridize with any of the known samples), the DNA amplified from the original sample could be used to determine the nucleotide sequence of the novel serosubtype and to clone the corresponding variable region into bacteriophage M13. It may be possible to develop this procedure for the diagnostic detection and typing of meningococci directly from clinical samples even when culture is not possible because of antibiotic treatment of an acute case.

M protein gene typing of Streptococcus pyogenes by nonradioactively labeled oligonucleotide probes.

Abstract: A new approach for the typing of Streptococcus pyogenes is described. Oligonucleotide probes of 30 nucleotides in length were derived from currently known sequences of the N-terminal regions of M protein genes (emm genes). The oligonucleotides were labeled with digoxigenin-dUTP and hybridized to dot-blotted genomic DNA from 116 group A streptococcal strains of serotypes M-1, M-2, M-3, M-5, M-6, M-12, M-18, M-19, M-24, and M-49. Hybridization reactions were visualized with a chemiluminescent substrate. In comparison with conventional serological typing of expressed M proteins, the binding of the probes to the corresponding emm genes exhibited 100% sensitivity and specificity. The results emphasize the high degree of type-specific conservation of the N-terminal regions of emm genes from reference strains and epidemiologically unrelated U.S. and European clinical isolates. The existence of two distinct genetic subgroups among eight investigated M-49 strains was unequivocally shown by hybridization assays and further confirmed by nucleotide sequence data obtained from four selected M-49 strains. Because oligonucleotide probes are relatively easy to prepare, easy to handle, and known to give consistent interlaboratory results, the "oligotyping" technique appears to offer potential advantages over conventional serological typing methods.

An epidemic methicillin-resistant strain of Staphylococcus aureus in Spain.

Abstract: During 1990, a strain of methicillin-resistant Staphylococcus aureus became epidemic in Spain and spread in a manner analogous to that of EMRSA-1 in England. Isolates of this strain produced little protein A and were resistant to a number of antibiotics including ciprofloxacin. Beta-lactamase production was encoded by a c. 39 kb plasmid, which also conferred resistance to mercury, cadmium, ethidium bromide and propamidine isethionate. Investigation showed that two variants, separable by supplementary and Fisk phage typing, were circulating. The B variant appeared to spread more readily than the A variant. The opportunity was taken to compare the discriminatory power of traditional typing methods with molecular techniques. The discriminatory power of the molecular techniques used only reached the same level as the traditional methods when double enzyme digestion of total cellular DNA by EcoR I and Cla I was performed.

Latex agglutination-negative methicillin-resistant Staphylococcus aureus recovered from neonates: epidemiologic features and comparison of typing methods.

Abstract: An unusual strain of methicillin-resistant Staphylococcus aureus (MRSA) was repeatedly isolated from infants in a newborn special care unit (NBSC) and a newborn intensive care unit. Between January 1989 and March 1990, approximately 100 isolates from infected or colonized infants were recovered. Surveillance cultures taken during this time revealed a 20% colonization rate, which was defined as recovery of MRSA from the nares, umbilicus, or groin. Isolates were identified as S. aureus by tube coagulase reactivity and heat-stable nuclease production but were unreactive in a latex agglutination assay. Representative isolates that were collected during the outbreak and that were found to share the latex agglutination assay-negative phenotype were compared by antibiogram (12 isolates), bacteriophage typing (20 isolates), capsular polysaccharide typing (30 isolates), and plasmid as well as chromosomal DNA analyses (20 isolates). All isolates known to be associated with the outbreak had nearly identical antibiograms and were notably susceptible to clindamycin. Staphylococcal bacteriophage typing was not useful in determining the relatedness of the isolates, since the majority were nontypeable. Plasmid pattern analysis revealed one large plasmid (approximately 100 kb) of equivalent size among the isolates. Capsular polysaccharide typing revealed that 14 of 30 isolates tested were type 5. Isolates identified in children at two other hospitals in the city which were also unreactive by the latex agglutination assay and clindamycin susceptible had plasmid and antibiogram patterns identical to those of isolates from the NBSC. Pulsed-field gel electrophoresis of restriction enzyme-digested genomic DNAs from the outbreak isolates demonstrated identical patterns which could be clearly differentiated from those of other unrelated MRSA. The strain from the NBSC is, therefore, unique and underscores the need for caution in interpreting the latex agglutination reactivities of MRSA isolates.

A new source of polymorphic DNA markers for sperm typing: analysis of microsatellite repeats in single cells.

Abstract: We show that dinucleotide and tetranucleotide repeat polymorphisms can be analyzed in single cells without using radioactivity or denaturing gels. This provides a rich new source of DNA polymorphisms for genetic mapping by sperm typing. The recombination fraction between two CA repeat polymorphisms was determined after whole genome amplification of single sperm, followed by typing of two different aliquots, one aliquot for each polymorphic locus. Single-cell analysis of microsatellites may also be valuable both for preimplantation genetic disease diagnosis based on single-blastomere or polar-body analysis and for the typing of forensic or ancient DNA samples containing very small amounts of nucleic acid.

Ribosomal RNA gene restriction patterns of Helicobacter pylori: analysis and appraisal of Hae III digests as a molecular typing system.

Abstract: Ribosomal RNA gene restriction patterns (ribopatterns) of 162 strains of Helicobacter pylori from 93 patients were studied to assess their suitability for use as the basis of a molecular typing system. Computer-assisted numerical analysis of Hae III ribopatterns of 122 strains from 9 countries in 4 continents showed that almost every strain had a distinct and unique ribopattern and only strains from the same individual were genomically matched in all bands or with minor (1-2 band) differences. Hae III ribopatterns offered high typability and reproducibility but were too discriminatory for large-scale epidemiological typing purposes because no rational basis for grouping strains was evident. In contrast, composite band profiles based on 21 band loci within the Hae III ribopatterns, which were used to compare strain sets selected on toxigenicity and geographical origin, were more conserved. Minor differences between some strain sets in several band loci were detected but generally the composite profiles were a reproducible feature of H. pylori. We conclude that Hae III ribopatterns provide an excellent fingerprint for small-scale studies of genomic variation in defined populations, such as sequential patient isolates, but are too specific for general typing of H. pylori.

Gene typing of Chlamydia trachomatis by polymerase chain reaction and restriction endonuclease digestion.

Abstract: A portion of the major outer membrane protein (MOMP) gene from 15 Chlamydia trachomatis serovars was amplified by polymerase chain reaction (PCR) and the product was analyzed by restriction fragment length polymorphism (RFLP). A set of primers was used to amplify an 871 base pair gene fragment encompassing the 4 hypervariable regions of MOMP. AluI digestion of the product gave distinctive patterns for the 15 serovars as demonstrated on silver-stained polyacrylamide gels. A triple digest with EcoRI, HinfI, and HpaII allowed improved discrimination between closely related serovars (C, H, I, J, L3). PCR and RFLP were used to type 50 wild-type clinical isolates and results were compared to results of the solid-phase enzyme immunoassay typing method. These isolates represented the most prevalent genital serovars (D, E, F, K, I and J) in the local sexually transmitted diseases clinic population. For specimens containing 1 serovar, the results of the two methods were similar for 42 samples and discordant for 1 sample. In addition, two samples showed evidence of mixed infection with two serovars as identified by both methods. Five additional specimens contained two serovars, as shown by one or both methods. In all five such specimens, the two typing methods agreed on at least one of the two serovars. For both single and multiple serovar specimens, there was concordance between the two typing methods for 16/17 E serovars, 8/9 D serovars, 8/8 F serovars, 7/7 I serovars, 7/7 J serovars, 5/8 K serovars, and 0/2 G serovars.(ABSTRACT TRUNCATED AT 250 WORDS)

HLA-DQB1 allele typing by a new PCR-RFLP method: correlation with a PCR-SSO method.

Abstract: We have developed a new PCR-RFLP method for HLA-DQB1 typing. This method was easy to follow, requiring only one DQB1 generic amplification and 5 endonucleases to assign 14 out of 15 HLA-DQB1 alleles. In addition, we determined that by using one generic amplification and two enzymes (Sau96 I and Hae III) it was possible to type the generic specificities: DQw2, DQw4, DQw5, DQw6, and DQw7, DQw8-9, providing a practical alternative for serological HLA-DQ generic typing. We also performed a side-by-side correlation with a PCR-SSO typing method and found an almost 100% concordance between the methods. The limitations of these methods were: 1) the PCR-RFLP method did not allow the differentiation between the HLA-DQB1*0602 and *0603 alleles; 2) the PCR-SSO method gave crosshybridization signals in the detection of *0302 or *0303 alleles. Our results suggested that both methods, PCR-RFLP and PCR-SSO, are useful alternatives for HLA-DQB1 typing.

DNA typing of isolates associated with the 1988 sporotrichosis epidemic.

Abstract: DNA typing techniques were used to examine selected clinical and environmental isolates of Sporothorix spp. recovered from the 1988 sporotrichosis epidemic in multiple states of the United States. Previous studies indicated that isolates in one of the six morphologically or physiologically distinct groups (group I) obtained from environmental sources were Sporothrix schenckii and were the possible etiologic agents responsible for the epidemic. To assess this hypothesis at the genetic level, whole-cell DNA was extracted from selected clinical isolates and representative members of each of the six environmental groups subjected to endonuclease digestion and then analyzed by gel electrophresis. DNA types were assigned on the basis of restriction fragment length polymorphism patterns. One DNA type was common to clinical and group I isolates but was dissimilar from the DNA types exhibited by groups II to VI. In contrast, a variety of DNA types were associated with isolates in groups II to VI. The latter groups appeared to make up a heterogeneous collection of fungi, with some members of the same group having different DNA types but with others from different groups possessing identical DNA types. Thus, DNA typing studies confirmed that group I environmental isolates are indistinguishable from clinical isolates and that group II to VI isolates represent a complex of related fungi with similar Sporothrix anamorphs. Images

Typing of Listeria monocytogenes by restriction polymorphism of the ribosomal ribonucleic acid gene region

Abstract: Summary Ninety four strains of Listeria monocytogenes of different serovars and phagovars as well as of varying origins were characterized by ribosomal RNA gene restriction polymorphism. After digestion by Eco RI or Hind III, chromosomal DNAs were hybridized with a cloned rDNA probe from Bacillus subtilis that included the 16S rRNA gene. The 94 strains were divided into 14 ribovars according to the different hybridization patterns generated by cleavage with Eco RI. Less important genomic heterogeneity could be detected when DNAs were digested by Hind III. Eco RI ribovars analysis allowed to describe a new typing scheme which did not corroborate routine typings such as serotyping and phage typing. It also confirmed a new view of this species in exhibiting a clone gathering most human strains, as first inferred from multilocus enzyme analysis ( Piffaretti et al., 22).