Fluorescence in situ hybridization (FISH) for direct visualization of microorganisms.

Fluorescence in situ hybridisation for the identification and characterisation of prokaryotes.

http://n.ethz.ch/~nbennett/download/Reading_NEW/DNA%20recognition/Nielsen.pdf

Peptide nucleic acid: a versatile tool in genetic diagnostics and molecular biology.

PNA for rapid microbiology.

A new fluorescence in situ hybridization (FISH) method with peptide nucleic acid (PNA) probes for identification of Staphylococcus aureus directly from positive blood culture bottles that contain gram-positive cocci in clusters (GPCC) is described. The test (the S. aureus PNA FISH assay) is based on a fluorescein-labeled PNA probe that targets a species-specific sequence of the 16S rRNA of S. aureus. Evaluations with 17 reference strains and 48 clinical isolates, including methicillin-resistant and methicillin-susceptible S. aureus species, coagulase-negative Staphylococcus species, and other clinically relevant and phylogenetically related bacteria and yeast species, showed that the assay had 100% sensitivity and 96% specificity. Clinical trials with 87 blood cultures positive for GPCC correctly identified 36 of 37 (97%) of the S. aureus-positive cultures identified by standard microbiological methods. The positive and negative predictive values were 100 and 98%, respectively. It is concluded that this rapid method (2.5 h) for identification of S. aureus directly from blood culture bottles that contain GPCC offers important information for optimal antibiotic therapy.

https://jcm.asm.org/content/jcm/40/1/247.full.pdf

Rapid Identification of Staphylococcus aureus Directly from Blood Cultures by Fluorescence In Situ Hybridization with Peptide Nucleic Acid Probes

TB PNA FISH is a new fluorescence in situ hybridization (FISH) method using peptide nucleic acid (PNA) probes for differentiation between species of the Mycobacterium tuberculosis complex (MTC) and nontuberculous mycobacteria (NTM) in acid-fast bacillus-positive (AFB+) cultures is described. The test is based on fluorescein-labelled PNA probes that target the rRNA of MTC or NTM species applied to smears of AFB+ cultures for microscopic examination. Parallel testing with the two probes serves as an internal control for each sample such that a valid test result is based on one positive and one negative reaction. TB PNA FISH was evaluated with 30 AFB+ cultures from Denmark and 42 AFB+ cultures from Thailand. The MTC-specific PNA probe showed diagnostic sensitivities of 84 and 97%, respectively, and a diagnostic specificity of 100% in both studies, whereas the NTM-specific PNA probe showed diagnostic sensitivities of 91 and 64%, respectively, and a diagnostic specificity of 100% in both studies. The low sensitivity of the NTM-specific PNA probe in the Thai study was due to a relatively high prevalence of Mycobacterium fortuitum, which is not identified by the probe. In total, 63 (87%) of the cultures were correctly identified as MTC (n = 46) or NTM (n = 17), whereas the remaining 9 were negative with both probes and thus the results were inconclusive. None of the samples were incorrectly identified as MTC or NTM; thus, the predictive value of a valid test result obtained with TB PNA FISH was 100%.

Fluorescence In Situ Hybridization Assay Using Peptide Nucleic Acid Probes for Differentiation between Tuberculous and Nontuberculous Mycobacterium Species in Smears of Mycobacterium Cultures

Novel hybridisation assay probes and mixtures of such probes for detecting a target sequence of one or more mycobacteria optionally present in a sample. The probes may suitably be directed to target sequences of mycobacterial rDNA, precursor rRNA, or rRNA, said probes being capable of forming detectable hybrids. The probes are in particular directed to mycobacterial rDNA, to precursor rRNA, or to 23S, 16S or 5S rRNA. The probes are useful for detecting the organisms in test samples such as sputum, laryngeal swabs, gastric lavage, bronchial washings, biopsies, aspirates, expectorates, body fluids (spinal, pleural, pericardial, synovial, blood, pus, bone marrow), urine, tissue sections as well as food samples, soil, air and water samples, and cultures thereof.

Novel probes for the detection of mycobacteria

Novel hybridisation assay probes and mixtures of such probes for detecting a target sequence of one or mycobacteria optionally present in a sample. The probes may suitable be directed to target sequences of mycobacterial rDNA, precursor rRNA or rRNA, said probes being capable of forming detectable hybrids. The probes are in particular directed to mycobacterial rDNA, to precursor rRNA, or to 23S, 16S or 5S rRNA. The probes are useful for detecting the organisms in test samples such as sputum, laryngeal swabs, gastric lavage, bronchial washings, biopsies, aspirates, expectorates, body fluids (spinal, pleural, pericardial synovial, blood, pus, bone marrow), urine, tissue sections as well as food samples, soil, air and water samples, and cultures thereof.

Kaare Lund

Papers

Fluorescence In Situ Hybridization Assay Using Peptide Nucleic Acid Probes for Differentiation between Tuberculous and Nontuberculous Mycobacterium Species in Smears of Mycobacterium Cultures

Novel probes for the detection of mycobacteria

Probes for the detection of mycobacteria