Critical assessment of staining properties of a new visualization technology: a novel, rapid and powerful immunohistochemical detection approach.

Immunohistochemical staining of tissue sections is a vital technique in pathological diagnostics and theranostics. Several kinds of detection systems are available-each of them with their advantages and disadvantages. Here we present the results of a study assessing a prototype immunohistochemical detection technology (PIDT) for visualization of antigens in tissue sections. Different tumor tissues (n = 11) were stained with selected antibodies (n = 30) and a subset of these under different fixation conditions. The staining properties were assessed according to six staining quality parameters (signal distribution, intensity, tissue and slide background, acutance, clarity of details, and subcellular morphological details), and the results were compared with those of a well-established detection system (EnVision FLEX). Overall, both detection methods revealed good to optimal results regarding the evaluated parameters even under unfavorable fixation conditions. However, with the prototype detection technology a quicker turnaround time was reached primarily due to shorter primary antibody incubation times. Moreover, PIDT-stained tissues showed higher signal intensity and a uniform signal distribution over the tissue slide, still, with well-preserved tissue morphology and without impairing the gradation of staining intensity of different cell types. In particular, the prototype detection technology performed better in poorly or delayed fixed tissue. In situations where rapid and profound results are in demand, and particularly in the context of a small laboratory setting, this prototype detection technology could be a useful addition to the established detection systems.

Evaluation of a fluorescence in situ hybridization assay for differentiation between tuberculous and nontuberculous Mycobacterium species in smears of Lowenstein-Jensen and Mycobacteria Growth Indicator Tube cultures using peptide nucleic acid probes.

A new fluorescence in situ hybridization assay based on peptide nucleic acid probes (MTB and NTM probes targeting tuberculous and nontuberculous species, respectively) for the identification of Mycobacterium tuberculosis complex and differentiation between tuberculous and nontuberculous mycobacteria (NTM) was evaluated using Lowenstein-Jensen (LJ) solid cultures from 100 consecutive sputum samples and 50 acid-fast bacillus (AFB)-positive sputum samples as well as Mycobacteria Growth Indicator Tube (MGIT) liquid cultures from 80 AFB-positive sputum samples. Mycobacterium species could be identified from a total of 53 LJ cultures and 77 MGIT cultures. The diagnostic specificities of the MTB and NTM probes were 100% for both cultures. The diagnostic sensitivities of the MTB probe for the LJ and MGIT cultures were 98 and 99%, respectively, whereas the sensitivities of the NTM probe were 57 and 100%, respectively. The relatively low sensitivity of the NTM probe was due to a high proportion of M. fortuitum, which is not identified by the probe.

Evaluation of a fluorescence hybridisation assay using peptide nucleic acid probes for identification and differentiation of tuberculous and non-tuberculous mycobacteria in liquid cultures.

The performance was evaluated of a fluorescence in situ hybridisation assay using peptide nucleic acid probes (Dako Probe MTB Culture Confirmation Test; Dako, Denmark) for identification of Mycobacterium tuberculosis complex (MTC) organisms and differentiation between tuberculous and non-tuberculous mycobacteria (NTM) in material taken directly from Bactec 12B (Becton Dickinson, USA) and MB/BacT (Organon Teknika, USA) bottles. The test was applied to 129 smear-positive (Ziehl-Neelsen stain) clinical specimens, 48 previously identified clinical strains of mycobacteria (12 MTC and 36 NTM), and 51 reference strains (7 MTC and 44 NTM) which were all previously inoculated into Bactec 12B and MB/BacT bottles. The sensitivity and specificity of the assay for MTC-positive cultures was 87.6% and 100%, respectively, for Bactec 12B, and 100%, respectively, for MB/BacT. The sensitivity and specificity of the assay for NTM-positive cultures was 100% for both media.

Fluorescence In situ hybridization assay using peptide nucleic acid probes for differentiation between tuberculous and nontuberculous mycobacterium species in smears of mycobacterium cultures.

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%.

Separation of Listeria monocytogenes and Salmonella berta from a complex food matrix by aqueous polymer two-phase partitioning.

In the present study, the use of aqueous polymer two-phase systems for separation of pathogenic bacteria from a complex food sample was investigated. Three different two-phase systems, a polyethylene glycol 3350/dextran T 500, a methoxy polyethylene glycol 5000/dextran T 500 and a polyethylene glycol 3350/hydroxypropyl starch system, were compared at pH 3 and pH 6 for their capacity to separate the pathogenic bacteria Listeria monocytogenes and Salmonella berta from a Cumberland sausage. In all three phase systems, the food particles partitioned to the lower phase. Best performance was obtained by the polymer combinations, polyethylene glycol 3350/dextran T 500 and polyethylene glycol 3350/hydroxypropyl starch. In these systems, Salmonella berta partitioned to the hydrophobic upper phase both at pH 3 and pH 6 with an average partitioning ratio of 80% and a recovery of 56%. Listeria monocytogenes partitioned to the upper phase at pH 3 only with an average partitioning ratio of 72% and a recovery of 45%. This method may become a valuable tool for separation of bacteria from complex food matrices.

Detection of Campylobacter jejuni and Camp. coli in chicken faecal samples by PCR.

PCR primers were selected from the flagellin gene sequences flaA and flaB of Campylobacter coli to amplify DNA from Camp. jejuni and Camp. coli. When the PCR products were analysed by hybridization to an internal probe immobilized in microtitre wells, positive reactions were observed only for strains of Camp. jejuni and Camp. coli. The assay was used to analyse 31 chicken faecal samples. Full correspondence was found between the PCR assay conducted on the enriched cultures and the standard culture method. When analysing the transport medium prior to enrichment, the PCR assay detected nine of 11 culture positive samples.

Listeria monocytogenes exists in at least three evolutionary lines: evidence from flagellin, invasive associated protein and listeriolysin O genes.

Regions of the genes encoding flagellin (flaA), the invasive associated protein (iap), listeriolysin O (hly) and 23S rRNA were sequenced for a range of Listeria monocytogenes isolates of different origin and serotypes. Several nucleotide sequence variations were found in the flaA, iap and hly genes. No differences were found for the rRNA genes, but our approach does not exclude the existence of differences between single copies of these genes. Based on the sequence differences, the L. monocytogenes strains can be divided into three distinct sequence types. Further, the presence of only a small number of sequence differences within each group indicates a strong degree of conservation within the groups. There was a complete correspondence among the groups of strains formed according to the analysis of the flaA, iap and hly genes, and the grouping correlates with serotype, pulsed field gel electrophoretic and multilocus enzyme electrophoretic data. Analysis of the region encoding the threonine-asparagine repeat units in the iap gene revealed some striking features. Sequence type 1 strains were found to have 16-17 repeats, sequence type 2 strains had 16-20 repeats whereas the two sequence type 3 strains analysed had only 11 repeats. Furthermore, within a 19 bp segment there was a 37% difference between the sequences of type 1 and 2 strains and that segment was absent in type 3 strains. Within the threonine-asparagine repeat region the nucleotide differences gave rise to four amino acid changes; however, all were changes among the three amino acids present in the repeat structure indicating a strong selective pressure on the composition of this region.

Detection of Yersinia enterocolitica O:3 in faecal samples and tonsil swabs from pigs using IMS and PCR.

Dilutions of faecal samples spiked with Yersinia enterocolitica O:3 were analysed using immunomagnetic separation (IMS) followed by PCR. In 10% faecal dilutions with added Y. enterocolitica cells, the limit of detection was 200 cells g-1 faeces. Faecal samples from 38 pigs were analysed by IMS-PCR in parallel with detection and quantification of Y. enterocolitica O:3 using cold pre-enrichment culturing. Of the 15 culture-positive samples, only two were detected with IMS-PCR. These two samples contained 40-400 Y. enterocolitica O:3 cells g-1 faeces; the highest level found in the investigation. This indicated that the low sensitivity of IMS-PCR was due to low amounts of cells in the faecal samples. Swab samples from 195 pig tonsils, taken on a slaughterline were examined using IMS-PCR and culture detection. Of 164 culture-positive samples, 60 were positive with IMS-PCR. In addition, IMS-PCR was positive for three culture-negative samples. Forty-five of the samples were further examined by IMS-PCR after 7-10 d of cold pre-enrichment. All 31 culture-positive samples as well as five culture-negative samples were detected by IMS-PCR. From these data it can be concluded that IMS-PCR can be used to detect Y. enterocolitica O:3 cells after pre-enrichment, but direct detection needs further optimization of the sample preparation procedures.

Oligonucleotide probes specific for the genus Salmonella and for Salm. typhimurium.

Synthetic oligonucleotides have been deduced and synthesized based on the sequence of a salmonella-specific polynucleotide probe. Two oligonucleotide probes, ST4 and ST15rev hybridized to 93 and 92 strains respectively out of 93 strains of Salmonella analysed. ST4, however, cross hybridized to one of the 28 strains of 16 genera of Enterobacteriaceae tested. Based on sequence alignment, in 16 strains of Salmonella, of a 114 base pair region, a Salm. typhimurium specific oligonucleotide probe, ST22, was identified. In colony hybridization, this probe detected all 47 strains of Salm. typhimurium analysed without hybridization to 94 strains of other Salmonella serotypes and to 26 strains of non-Salmonella bacteria.

RAPD analysis of Yersinia enterocolitica.

A total of 87 isolates of Yersinia enterocolitica were examined with randomly amplified polymorphic DNA (RAPD) by use of three different primers. Based on the RAPD profiles, the strains could be divided into three major groups: (1) the pathogenic American serotypes, O:8, O:13ab, O:20 and O:21; (2) the pathogenic European serotypes, O:3, O:5,27 and O:9; and (3) the nonpathogenic serotypes. Five tested strains of the American serotype O:4 gave unique profiles with YCPEL, but did not give reproducible profiles with the other primers. The European serotypes could be further subdivided into a group consisting of strains of O:3 and O:5,27 and a group of strains of O:9. RAPD profiling provides an easy approachable method to divide isolates of Y. enterocolitica into pathogenic and nonpathogenic strains and further to differentiate between the pathogenic isolates.

Analysis of fluorescent pseudomonads based on 23S ribosomal DNA sequences.

The regions from positions 1391 to 1545 and 1620 to 1865 (Escherichia coli numbers) of the 23S ribosomal DNA sequences have been analyzed for a number of Pseudomonas fluorescens and P. putida isolates. Variability was observed only in three smaller regions from positions 1484 to 1508, 1531 to 1542, and 1714 to 1748, corresponding to helices 58, 59, and 63, respectively, where up to 53% dissimilarity was found. Sequence analysis did not allow clear distinctions among P. fluorescens biovars, P. chlororaphis, and P. putida biovar B.

Specific detection of pathogenic Yersinia enterocolitica by two-step PCR using hot-start and DMSO.

A pair of polymerase chain reaction (PCR) primers, YC1 and YC2, selected from the sequence of the invasin locus (inv) of Y. enterocolitica, has been evaluated for specific detection of pathogenic Y. enterocolitica by PCR. The primers were hybridized at high stringency conditions to DNA from 65 pathogenic Y. enterocolitica, 16 non-pathogenic Y. enterocolitica, 18 other Yersinia and 124 non-Yersinia strains. YC2 hybridized to the pathogenic Y. enterocolitica only, while YC1 hybridized weakly to nine non-Yersinia as well. In a PCR with annealing at 64 degrees C all Y. enterocolitica, pathogenic and non-pathogenic, were positive. However, DNA from 60 non-Y. enterocolitica was amplified. With annealing at 72 degrees C, 10 non-pathogenic Y. enterocolitica and 41 non-Y. enterocolitica were positive. When a two-step PCR assay with annealing at 72 degrees C, hot-start and 1% dimethylsulphoxide (DMSO) were used, only DNA from the pathogenic Y. enterocolitica were amplified. The limit of detection was shown for four different strains to be less than 10 cells per PCR tube.

Characterization of two putative Listeria monocytogenes genes encoding polypeptides homologous to the sensor protein CheA and the response regulator CheY of chemotaxis.

The nucleotide sequence of a region located downstream of the Listeria monocytogenes flagellin gene, flaA, has been determined. DNA sequence analysis revealed the presence of two open reading frames with a potential to encode polypeptides of 13.1 and 68.7 kDa, respectively. The deduced polypeptides show a high degree of identity to the chemotactic proteins, CheY and CheA, respectively, from Bacillus subtilis and Escherichia coli. Moreover, significant features of CheY and CheA are conserved in the L. monocytogenes homologues. The high degree of conservation suggests that the polypeptides are involved in signal transduction controlling chemotaxis in L. monocytogenes and consequently the putative genes are named cheY and cheA. Northern blot and primer extension analysis suggested that cheY and cheA are transcribed as a bicistronic unit and that the transcription is thermoregulated.

Salmonella identification by the polymerase chain reaction.

Polymerase chain reaction (PCR) primers for genus specific detection of Salmonella have been selected from a Salmonella-specific fragment of 2.3 kilobases (kb). Due to interserovar sequence diversity within this fragment, primer selection was based on DNA sequence alignment of sequences from 20 different Salmonella serovars. The specific PCR product of 429 base pairs (bp) was formed from 144 of 146 salmonella strains tested (116 of 118 serovars). The two false-negative strains belonged to two different serovars of the rarely isolated subspecies IIIa (monophasic S. arizonae). No product was produced in any of 86 non-Salmonella Enterobacteriacea strains tested, covering 41 species from 21 genera.

A highly sensitive and fast nonradioactive method for detection of polymerase chain reaction products.

A novel system for the detection of polymerase chain reaction (PCR) products has been developed. The system is based on a PCR in which one of the primers is biotinylated and digoxigenin-11-dUTP is incorporated during elongation. Biotinylated PCR products are captured on streptavidin-coated solid supports, and alkaline phosphatase conjugated to anti-digoxigenin antibody is subsequently bound to the incorporated digoxigenin. The detection may be obtained with colorimetric, fluorescent, or luminescent substrates for alkaline phosphatase. The detection system can be performed in microtiter plates allowing easy handling of multiple samples. The total assay time following the PCR is between 1 and 2 h dependent on the type of substrate and the type of solid support applied in the system. Within this period of time the system is capable of detecting 1 template in 29 cycles of PCR.

Cloning and characterization of a gene encoding flagellin of Listeria monocytogenes.

The gene, flaA, encoding the flagellin protein of Listeria monocytogenes (strain 12067) has been isolated from an expression library in Escherichia coli using a flagellin-specific monoclonal antibody. DNA sequence analysis of a positive clone revealed the presence of an open reading frame of 287 amino acid residues with a calculated molecular mass of 30.4 kDa. Comparison of this sequence with flagellins from other bacteria showed a significant degree of homology in both the N- and C-terminal parts of the protein. The flagellin mRNA was determined to be 1 kb in size, which is the expected size for a monocistronic mRNA, and the temperature-dependent expression of flagellin was found to be regulated at the transcriptional level. Southern blot analysis, using the flagellin gene as probe, indicated that L. monocytogenes can be divided into two groups. These groups correspond to the flagellar antigens AB and ABC, respectively, as well as to the two types of L. monocytogenes based on the DNA sequence of the listeriolysin gene.

Inhibition of PCR by components of food samples, microbial diagnostic assays and DNA-extraction solutions.

We have tested the influence on the polymerase chain reaction (PCR) of a large number of compounds found in food, in media used for selective propagation of food-borne pathogens or in DNA-extraction methods. PCR was found to be sensitive to large volumes of complex food samples containing high amounts of fat and protein, however, an extraction procedure based on treatment with hot NaOH/SDS reduced the effect significantly. Some culture media (Fraser, MLEB, MRB and Rappaport) interfered with the analysis and for most of the media it was possible to assign the inhibitory effect to one or more individual components. Several compounds (detergents, lysozyme, NaOH, alcohols, EDGA, EGTA) used in DNA extraction procedures were found to have some inhibitory effect. The inhibitory effects need to be taken into consideration when designing new tests.

Nucleotide sequence, organization and characterization of the atp genes and the encoded subunits of Mycoplasma gallisepticum ATPase.

The nucleotide sequence of a 7.8 kbp DNA fragment from the genome of Mycoplasma gallisepticum has been determined. The fragment contains a cluster of nine tightly linked genes coding for the subunits of the M. gallisepticum ATPase. The gene order is I (I-subunit), B (a-subunit), E (c-subunit), F (b-subunit), H (delta-subunit), A (alpha-subunit), G (gamma-subunit), D (beta-subunit) and C (epsilon-subunit). Two open reading frames were identified in the flanking regions; one (ORFU), preceding the I gene, encodes at least 110 amino acids and the other (ORFS), following the C gene, encodes at least 90 amino acids. The deduced amino acid sequences of the various subunits are presented and discussed with regard to the structure, function and differing sensitivity of the M. gallisepticum enzyme to dicyclohexylcarbodiimide and aurovertin. The alpha- and beta-subunits of the F1 portion are well conserved (51% and 65% identity with those of Escherichia coli), whereas the gamma-, delta- and epsilon-subunits, as well as the F0-subunits, show a low percentage identity. Nonetheless, the secondary structure of the F0-subunits show a high degree of similarity to the corresponding subunits of E. coli. Two very strong potential amphipathic alpha-helices are predicted in the delta-subunit and the N-terminus of the b-subunit contains two hydrophobic helical stretches. The possible roles of these structural properties in the close association of the F1 and F0 multisubunit complexes among mycoplasmas are discussed.

Development and application of a new method to extract bacterial DNA from soil based on separation of bacteria from soil with cation-exchange resin.

A new method for the extraction of bacterial DNA from soil has been developed. Soil samples of 50 g were dispersed, and bacteria were released by use of a cation-exchange resin; subsequently, bacteria were separated from soil particles by low-speed centrifugation and lysed with lysozyme and ionic detergent, and the DNA was then purified by CsCl-ethidium bromide equilibrium density centrifugation. The extracted DNA was of high molecular weight and sufficiently pure for restriction enzyme digestion, DNA-DNA hybridization, and amplification by the polymerase chain reaction. The advantages of the new method are that the separation of bacteria from soil is considerably faster than by repeated blending, more samples can be handled, and furthermore no aerosols are formed during separation. Also, we investigated whether the CsCl-ethidium bromide equilibrium density centrifugation could be replaced by purification using Gene-Clean. However, this method produced DNAs which were insufficiently pure for several types of analysis. The new method was used to study survival of a 2,4-dichlorophenoxyacetic acid (2,4-D)-degrading Pseudomonas cepacia DBO1 (pRO101) in unamended soil and in soil amended with 2,4-D. We found that the degrading strain, irrespective of inoculation level, was able to grow to the same high numbers in soil amended with 2,4-D, while the strain in nonamended soil were maintained at the inoculation level. Detection based on DNA extraction and subsequent dot blot DNA-DNA hybridization was in accordance with detection by plating on selective medium.

Expression in E. coli of the gene encoding phenylalanine ammonia-lyase from Rhodosporidium toruloides.

The active sites of the enzyme phenylalanine ammonia-lyase (Pal) from Rhodosporidium toruloides contains a dehydroalanine residue that is believed to be essential for catalytic activity. Furthermore, the dehydroalanine is believed to be added post-translationally as part of a prosthetic group covalently attached to the enzyme. Perhaps for this reason no attempts to produce Pal in foreign host cells have been reported. We have inserted the entire uninterupted pal gene from R. toruloides into the Escherichia coli expression vector pKK 223-3. E. coli cells containing this vector synthesize a protein of the expected size, and extracts prepared from these cells contain a Pal-like activity. The potential implications of this finding are discussed.