Direct detection and identification of African trypanosomes by fluorescence in situ hybridization with peptide nucleic acid probes.

We have developed a rapid and easy to perform fluorescence in situ hybridization test that allows specific identification of trypanosomes from the subgenus Trypanozoon, using peptide nucleic acid probes. Probes were designed to target subgenus-specific sequences on the multiple-copy 18S rRNA, greatly facilitating the detection of a single trypanosome.

Identification of indicator microorganisms using a standardized PNA FISH method.

A standardized fluorescent in situ hybridization (FISH) method using Peptide Nucleic Acid (PNA) probes for analysis of gram-negative and gram-positive bacteria, as well as yeast, has been developed. Fluorescently labeled PNA probes targeting specific rRNA sequences of Escherichia coli, Pseudomonas aeruginosa, Staphyloccocus aureus, Salmonella were designed, as well as PNA probes targeting eubacteria and eucarya. These PNA probes were evaluated by PNA FISH using 27 bacterial and 1 yeast species, representing both phylogenetically closely related species, as well as species important to both clinical and industrial settings. The S. aureus and P. aeruginosa PNA probes did not cross react with any of the organisms tested, whereas the E. coli PNA probe, as expected from sequence data, also detected Shigella species. The Salmonella PNA probe reacted with all of the 13 Salmonella strains, representing the 7 subspecies of Salmonella, however, it is also complementary to a few other bacterial species. The eubacteria- and eucarya-specific PNA probes detected all bacterial species and one yeast species, respectively. The general applicability of the PNA FISH method made simultaneous identification of multiple species, both gram-negative and gram-positive, in a mixed population an attractive possibility never accomplished using DNA probes. Four color images using differently labeled PNA probes showed simultaneous identification of E. coli, P. aeruginosa, S. aureus and Salmonella, thereby demonstrating the potential of multiplex FISH for various diagnostic applications within both clinical and industrial microbiology.

Differentiation of Candida albicans and Candida dubliniensis by fluorescent in situ hybridization with peptide nucleic acid probes.

The recent discovery of Candida dubliniensis as a separate species that traditionally has been identified as Candida albicans has led to the development of a variety of biochemical and molecular methods for the differentiation of these two pathogenic yeasts. rRNA sequences are well-established phylogenetic markers, and probes targeting species-specific rRNA sequences have been used in diagnostic assays for the detection and identification of microorganisms. Peptide nucleic acid (PNA) is a DNA mimic with improved hybridization characteristics, and the neutral backbone of PNA probes offers significant advantages in whole-cell in situ hybridization assays. In this study, we developed PNA probes targeting the rRNAs of C. albicans and C. dubliniensis and applied them to a fluorescence in situ hybridization method (PNA FISH) for differentiation between C. albicans and C. dubliniensis. Liquid cultures were smeared onto microscope slides, heat fixed, and then hybridized for 30 min. Unhybridized PNA probe was removed by washing, and smears were examined by fluorescence microscopy. Evaluation of the PNA FISH method using smears of 79 C. dubliniensis and 70 C. albicans strains showed 100% sensitivity and 100% specificity for both PNA probes. We concluded that PNA FISH is a powerful tool for the differentiation of C. albicans and C. dubliniensis.

Self-reporting PNA/DNA primers for PCR analysis.

We report a new fluorogenic method for sealed-tube PCR analysis using a quencher-labeled peptide nucleic acid (Q-PNA) probe. The Q-PNA hybridizes to a complementary tag sequence located at the 5' end of a 5' fluorophore-labeled oligonucleotide primer, quenching the primer's fluorescence. Incorporation of the primer into a doublestranded amplicon causes displacement of the Q-PNA such that the fluorescence of the sample is a direct indication of the amplicon concentration. The Q-PNA is able to quench multiple primers bearing distinct 5' fluorophores in a single reaction. We show realtime quantitative detection of a single-copy gene, K-ras, from human genomic DNA, as well as an endpoint multiplex assay for Chlamydia trachomatis and Neisseria gonorrhoeae targets. Because the Q-PNA may be used to quench any primer that contains the 5' tag sequence, it is possible to inexpensively adapt an existing primer set for use in a self-reporting fluorescent assay by including the tag sequence in one of the primers.

Identification of Dekkera bruxellensis (Brettanomyces) from wine by fluorescence in situ hybridization using peptide nucleic acid probes.

A new fluorescence in situ hybridization method using peptide nucleic acid (PNA) probes for identification of Brettanomyces is described. The test is based on fluorescein-labeled PNA probes targeting a species-specific sequence of the rRNA of Dekkera bruxellensis. The PNA probes were applied to smears of colonies, and results were interpreted by fluorescence microscopy. The results obtained from testing 127 different yeast strains, including 78 Brettanomyces isolates from wine, show that the spoilage organism Brettanomyces belongs to the species D. bruxellensis and that the new method is able to identify Brettanomyces (D. bruxellensis) with 100% sensitivity and 100% specificity.

Filter-based PNA in situ hybridization for rapid detection, identification and enumeration of specific micro-organisms.

AIMS: A method for rapid and simultaneous detection, identification and enumeration of specific micro-organisms using Peptide Nucleic Acid (PNA) probes is presented. METHODS AND RESULTS: The method is based on a membrane filtration technique. The membrane filter was incubated for a short period of time. The microcolonies were analysed by in situ hybridization, using peroxidase-labelled PNA probes targeting a species-specific rRNA sequence, and visualized by a chemiluminescent reaction. Microcolonies were observed as small spots of light on film, thereby providing simultaneous detection, identification and enumeration. The method showed 95-100% correlation to standard plate counts along with definitive identification due to the specificity of the probe. CONCLUSION: Using the same protocol, results were generated approximately three times faster than culture methods for Gram-positive and -negative bacterial species and yeast species. SIGNIFICANCE AND IMPACT OF THE STUDY: The method is an improvement on the current membrane filtration technique, providing rapid determination of the level of specific pathogens, spoilage or indicator micro-organisms.

Rapid detection, identification, and enumeration of Escherichia coli cells in municipal water by chemiluminescent in situ hybridization.

A new chemiluminescent in situ hybridization (CISH) method provides simultaneous detection, identification, and enumeration of culturable Escherichia coli cells in 100 ml of municipal water within one working day. Following filtration and 5 h of growth on tryptic soy agar at 35 degrees C, individual microcolonies of E. coli were detected directly on a 47-mm-diameter membrane filter using soybean peroxidase-labeled peptide nucleic acid (PNA) probes targeting a species-specific sequence in E. coli 16S rRNA. Within each microcolony, hybridized, peroxidase-labeled PNA probe and chemiluminescent substrate generated light which was subsequently captured on film. Thus, each spot of light represented one microcolony of E. coli. Following probe selection based on 16S ribosomal DNA (rDNA) sequence alignments and sample matrix interference, the sensitivity and specificity of the probe Eco16S07C were determined by dot hybridization to RNA of eight bacterial species. Only the rRNA of E. coli and Pseudomonas aeruginosa were detected by Eco16S07C with the latter mismatch hybridization being eliminated by a PNA blocker probe targeting P. aeruginosa 16S rRNA. The sensitivity and specificity for the detection of E. coli by PNA CISH were then determined using 8 E. coli strains and 17 other bacterial species, including closely related species. No bacterial strains other than E. coli and Shigella spp. were detected, which is in accordance with 16S rDNA sequence information. Furthermore, the enumeration of microcolonies of E. coli represented by spots of light correlated 92 to 95% with visible colonies following overnight incubation. PNA CISH employs traditional membrane filtration and culturing techniques while providing the added sensitivity and specificity of PNA probes in order to yield faster and more definitive results.

Rapid detection, identification, and enumeration of Pseudomonas aeruginosa in bottled water using peptide nucleic acid probes.

A new chemiluminescent in situ hybridization (CISH) method that provides simultaneous detection, identification, and enumeration of Pseudomonas aeruginosa in bottled water within 1 working day has been developed. Individual micro-colonies of P. aeruginosa were detected directly on membrane filters following 5 h of growth by use of soybean peroxidase-labeled peptide nucleic acid (PNA) probes targeted to a species-specific sequence in P. aeruginosa rRNA. Within each micro-colony, reaction of the peroxidase with a chemiluminescent substrate generated light that was subsequently captured by film or with a digital camera system. Each spot of light represented one micro-colony of P. aeruginosa. Sensitivity and specificity for the identification of P. aeruginosa were 100% as determined by testing 28 P. aeruginosa strains and 17 other bacterial species that included closely related Pseudomonas species. Furthermore, the number of micro-colonies of P. aeruginosa represented by light spots correlated with counts of visible colonies following sustained growth. We conclude that PNA CISH speeds up traditional membrane filtration techniques and adds the specificity of PNA probe technology to generate fast and definitive results.

Investigation of immunosuppressive properties of inactivated human immunodeficiency virus and possible neutralization of this effect by some patient sera.

Retroviral infections are accompanied by immunosuppression in a variety of species. For feline leukemia virus, the immunosuppression has been ascribed to the transmembrane envelope protein, p15E, which suppresses the proliferative responses of cat, mouse, and human lymphocytes. A similar suppressive effect has been shown for a lysate of human immunodeficiency virus (HIV), strain HTLV-IIIB. Here we determined that detergent-disrupted HTLV-IIIB lystate exerted a strong suppressive effect on PHA-stimulated lymphocytes. Preparations of whole virions, a lysate of a local HIV isolate grown on MP-6 cells, and a commercially obtained UV and psoralene-inactivated lysate were examined and demonstrated to have a similar suppressive effect. The HIV lysate was not directly cytotoxic to lymphocytes and did not contain tumor necrosis factor or lymphotoxin. The HIV lysate specifically suppressed the proliferation of a range of hemopoietic cell lines from man and mouse including three EBV transformed CD4- and IL-2 receptor-negative B-cell lines. The lysate also suppressed the formation of human bone marrow colonies, whereas the lysate had only a slight or no effect on fibroblasts. The suppression of lymphocyte proliferation was not abrogated by addition of IL-2 or IL-1 and the HIV lysate inhibited the expression of IL-2 receptors on suboptimal PHA-stimulated mononuclear cells. The suppressive factor(s) has not been characterized in molecular terms, but suppressive activity was recovered in fractions with a molecular weight of about 67,000 and in both the glycoprotein fraction and in the glycoprotein-depleted fraction of the HIV lysate. Sera from one-third of a small series (N = 13) of individuals with antibodies to HIV seem to be able to neutralize the suppressive properties of HIV lysate in cultures.

Segregation of HLA-DR, -DQ and -DP phenotypes and restriction fragment length polymorphic fragments in two recombinant families.

Members of two families were typed for HLA-DR, -DQ and -DP specificities by means of sera and local PLT bulk reagents. One B:C and one DR:DP cross-over were identified in both families. The restriction fragment length polymorphism (RFLP) was analyzed by southern blotting and the use of DR beta, DR alpha, DQ beta, DQ alpha, DP beta and DP alpha probes. Previous observations concerning typical RFLP patterns for serological DR/DQ- and cellular DP-specificities were confirmed. With respect to recombinant haplotypes, RFLP was informative in both families. All polymorphic fragments-(DRB, DQA and B as well as DPA and B)-segregated together in the HLA-B:DR cross-overs. In the DR:DP recombinant children, the DR and DQ fragments were separated from DP fragments, demonstrating that DP-types as defined by PLT and RFLP map close together. No cross-hybridization between the segregating fragments was detected with the various probes.

HLA-DP antigens are involved in the susceptibility to multiple sclerosis.

Forty-five unrelated patients with multiple sclerosis (MS) from Sweden and 166 Danish controls were typed for HLA-DP using Primed Lymphocyte Typing. Thirty-nine MS-patients and 63 controls were also DNA-typed with the Restriction Fragment Length Polymorphism (RFLP) technique for HLA-DP and -DR genes. The frequencies of DPw4 were 93.3% in MS patients and 72.3% in controls (relative risk, RR = 5.4, p = 0.0014). The DR2 antigen was present in 75.5% of the patients and in 33.7% of the controls (RR = 6.1, p less than 10(-6)). DPw4 was not associated (i.e., was not in linkage disequilibrium) with DR2 in patients or controls. Thus, in MS the associations with DP and DR are independent of each other. However, the combined presence of DPw4 and DR2 gave a significantly higher risk than each antigen alone, indicating that synergism between DP and DR gene products may play a role in the genetic susceptibility to MS.

Restriction fragment polymorphism (RFLP) of a "new" HLA-DP specificity, CDP-HEI.

Southern blotting with a DP beta cDNA probe of MspI digested DNA from 83 healthy unrelated individuals revealed a 1.8 kb fragment present in all four individuals (and no others) possessing the newly determined DP specificity, CDP-HEI.

Class II genes of the human major histocompatibility complex. Comparisons of the DQ and DX alpha and beta genes.

The human major histocompatibility complex, HLA, contains the genes of several class II molecules. We present here the molecular maps of the DQ and DX subregions and analyze the sequences of the polymorphic DQ alpha and DQ beta genes as well as the DX alpha and DX beta genes. The DQ alpha and DQ beta genes are oriented in opposite directions, approximately 12 kilobases apart. The DX alpha and DX beta genes are similarly oriented about 8 kilobases. The exon-intron organizations of the DQ alpha and DX alpha genes are analogous to those of other class II alpha genes. Comparison of the DQ alpha gene sequence to three DQ alpha cDNA clones shows that amino acid replacements are predominantly located between residues 45 and 80 in the amino-terminal domain. Analysis of the frequency of silent and replacement substitutions indicates that there is little selection against replacements in DQ alpha first domains. The exons encoding the second domains of DQ alpha and DX alpha are virtually identical, suggesting that a gene conversion event has occurred between these genes. The DX beta gene is very similar to the DQ beta gene but differs in the cytoplasmic portion. The DX beta gene contains a separate exon of 24 nucleotides encoding the core of the cytoplasmic tail. This exon is not expressed in the DQ beta genes due to a nonfunctional splice junction. Comparison of the number of nucleotide substitutions in the DQ beta first and second domain exons suggests that little or no phenotypic selection acts on the first domain whereas the second domain is under strong selection.

Restriction fragment length polymorphism of the HLA-DP subregion and correlations to HLA-DP phenotypes.

The restriction fragment length polymorphism (RFLP) of the class II HLA-DP subregion of the major histocompatibility complex (MHC) of humans has been unraveled by Southern blotting using DP alpha and DP beta probes in a study of 46 unrelated individuals with known HLA-DP types. Contrary to earlier preliminary findings with a limited number of enzymes, the RFLP appears to be quite extensive both with the DP beta (14 different DNA markers defined by individual fragments or clusters thereof) and the DP alpha (8 markers) probes, especially when enzymes recognizing only four base pairs were used. A few markers were absolutely or strongly associated with individual DP antigens, whereas most were associated with two or more DP antigens as defined by primed lymphocyte typing. Thus, Southern blotting seems feasible for typing for most DP determinants by specific fragments or subtraction between the various more broadly reactive DNA markers, and the RFLP provides further information on the DP subregion in addition to that provided by primed lymphocyte typing. In two recombinant families, the DP beta and DP alpha DNA markers segregated with DP antigens, whereas the DR beta, DQ beta, DQ alpha, and DX alpha markers followed the DR and DQ antigens.

A "new" supertypic HLA-DP related determinant detected by primed lymphocyte typing (PLT).

Primed Lymphocyte Typing (PLT) with local (CDP) and the 9th International Histocompatibility Workshop reagents (GNN) revealed "cross-reactions" between HLA-DPw6 and the GNN2B but not other DPw2 PLT-cells (GNN2A, CDP2A, CDP2B). We raised and bulk-expanded a well discriminating PLT-reagent (the JET-reagent) from an HLA-DR identical, DP, GNN2A, CDP2A, CDP2B compatible and GNN2B incompatible responder/stimulator combination. The JET-reagent defined a "new" determinant, JET, which was present in 11% of Danes. The JET determinant was associated with DPw2, w6, and DP-blank: 66% of DPw6 and DP-blank and 20% of DPw2-positive individuals were JET-positive. All JET-positive individuals belonged to this group, and six of these had two DP-antigens. JET segregated with DP-blank in three and with DPw6 in two informative families. In an HLA-DR/DPw6 recombinant family, JET segregated with DPw6 in the recombinant haplotype. The JET PLT-responses against all of six different JET-positive stimulators including the specific stimulator were strongly inhibited by the monoclonal antibody (MoAb) Tü39, which preferentially reacts with DP-molecules, but not by other HLA-specific MoAbs. The results indicate that JET is either a "new" supertypic DP-related specificity or a determinant, which shares epitopes with DP, of a "new" locus and in linkage disequilibrium with DP.

Increased frequency of HLA-DPw2 in pauciarticular onset juvenile chronic arthritis.

Thirty-six unrelated Danish patients with pauciarticular Juvenile Chronic Arthritis (PJCA) and 120 controls were typed for HLA-DPw1-w6 and the local specificity CDPHEI with bulk-expanded Primed Lymphocyte Typing (PLT) cells. The frequency of HLA-DPw2 was 52.8% in PJCA patients and 16.7% in controls (relative risk, RR = 4.5; P less than 0.001). The antigens HLA-Dw5 and/or Dw8 were present in 50% of the patients and in 21.3% of the controls (RR = 4.2; p less than 10(-3)). DPw2 was not associated (in linkage disequilibrium) with Dw5/w8 in patients or in controls, and the DP and D associations with PJCA were independent of each other. However, the combined presence of DPw2 and Dw5 and/or Dw8 gave a significantly higher risk of PJCA than each antigen alone indicating interaction of DP and DR gene products. PJCA is the first disease definitely found to be associated with a DP antigen.

Mutations and selection in the generation of class II histocompatibility antigen polymorphism.

A comparison of seven human DR and DC class II histocompatibility antigen beta-chain amino acid sequences indicates that the allelic variation is of comparable magnitude within the DR and DC beta-chain genes. Silent and replacement nucleotide substitutions in six DR and DC beta-chain sequences, as well as in seven murine class II sequences (three I-A beta and four I-A alpha alleles) were analyzed. The results suggest that the mutation rates are of a comparable magnitude in the nucleotide sequences encoding the first and second external domains of the class II molecules. Nevertheless, the allelic amino acid replacements are predominantly located in the first domains. We conclude that a conservative selective pressure acts on the second domains, whereas in many positions in the first domains replacement substitutions are selectively neutral or maybe even favoured. Thus, the difference between the first and second domains as regards the number of amino acid replacements is mainly due to selection.

Exon-intron organization and complete nucleotide sequence of a human major histocompatibility antigen DC beta gene.

We have determined the complete nucleotide sequence of a human class II histocompatibility antigen DC beta gene. The gene spans more than 7 kilobases and contains five exons corresponding to the different domains of the DC beta polypeptide. The exon-intron organization is thus analogous to that of class II antigen alpha-chain genes, class I antigen heavy chain genes, and the constant parts of immunoglobulin genes, emphasizing further the evolutionary relationship among these molecules. The mature polypeptide deduced from the DC beta gene shows 93% and 88% homology, respectively, to sequences derived from two DC beta cDNA clones of other haplotypes. The allelic polymorphism of DC beta chains resides predominantly in the first extracellular domain, whereas the rest of the polypeptide is virtually constant. The exons of the DC beta gene display high homology to the corresponding exons of a murine I-A beta gene. Also, the introns show significant homology. The DC beta chains lack eight amino acids in the cytoplasmic tail, as compared to DR and I-A beta chains. This is probably due to a nonfunctional splice junction of DC beta genes, causing a separate cytoplasmic exon to be nonexpressed.

The complete nucleotide sequence of the I-E alpha d immune response gene.

We have isolated and sequenced the complete murine I-E alpha immune response gene of the H-2db haplotype. The I-E alpha d gene consists of 5300 basepairs and is organized into five or possibly six exons that correspond to different domains of the alpha chain. The amino acid sequence deduced from the I-E alpha gene shows 75% homology to its human counterpart, the HLA-DR alpha chain. The absence of I-E antigen in H-2 mice is due to lack of E alpha chain synthesis. We show here that this defect is caused by a deletion in the 5' end of the I-E alpha b gene.