Amplification of RNA by NASBA allows direct detection of viable cells of Ralstonia solanacearum in potato

AIMS: The objective of this study was to develop a Nucleic Acid Sequence Based Amplification (NASBA) assay, targeting 16S rRNA sequences, for direct detection of viable cells of Ralstonia solanacearum, the causal organism of bacterial wilt. The presence of intact 16S rRNA is considered to be a useful indicator for viability, as a rapid degradation of this target molecule is found upon cell death. METHODS AND RESULTS: It was demonstrated by RNase treatment of extracted nucleic acids from R. solanacearum cell suspensions that NASBA exclusively detected RNA and not DNA. The ability of NASBA to assess viability was demonstrated in two sets of experiments. In the first experiment, viable and chlorine-killed cells of R. solanacearum were added to a potato tuber extract and tested in NASBA and PCR. In NASBA, only extracts spiked with viable cells resulted in a specific signal after Northern blot analysis, whereas in PCR, targeting 16S rDNA sequences, both extracts with viable and killed cells resulted in specific signals. In the second experiment, the survival of R. solanacearum on metal strips was studied using NASBA, PCR-amplification and dilution plating on the semiselective medium SMSA. A positive correlation was found between NASBA and dilution plating detecting culturable cells, whereas PCR-amplification resulted in positive reactions also long after cells were dead. The detection level of NASBA for R. solanacearum added to potato tuber extracts was determined at 104 cfu per ml of extract, equivalent to 100 cfu per reaction. With purified RNA a detection level of 104 rRNA molecules was found. This corresponds with less than one bacterial cell, assuming that a metabolically active cell contains ca 105 copies of rRNA. Preliminary experiments demonstrated the potential of NASBA to detect R. solanacearum in naturally infected potato tuber extracts. CONCLUSIONS: NASBA specifically amplifies RNA from viable cells of R. solanacearum even present in complex substrates at a level of 100 cfu per reaction. SIGNIFICANCE AND IMPACT OF THE STUDY: The novel NASBA assay will be particularly valuable for detection of R. solanacearum in ecological studies in which specifically viable cells should be determined.     

Hemopoietic stem and precursor cell analysis in umbilical cord blood using the Sysmex SE-9000 IMI channel

Circulating hematopoietic progenitor cells (HPCs) are routinely measured by flow cytometry using CD34 expression. As an alternative, the "immature information" (IMI) channel measurement of the automated hematology analyzer Sysmex SE machines was developed. We tested the IMI channel HPC method with umbilical cord blood specimens. The IMI-HPCs were compared with CD34 counts and numbers of colony-forming units (CFUs). The IMI-HPC data were reproducible and dilution experiments yielded a log-linear relationship. The mean percentage of CD34(+) cells in 50 umbilical cords was 0.43 versus 0.11 of HPCs in the IMI channel (correlation coefficient r = 0.67). Absolute numbers yielded 96.79 x 10(6)/L CD34(+), 33.17 x 10(6)/L IMI-HPC, and 35.04 x 10(6)/L CFU-HPC. Receiver operating characteristics curves were made at various cutoff levels for CD34(+) cells to visualize sensitivity and specificity profiles. With median values of 13.56 x 10(6)/L for IMI-HPC and 20 x 10(6)/L for CD34(+) as cutoff points (the levels used in the laboratory to start stem cell pheresis), the percentage of false-negative observations was 70.4%. To exclude the influence of storage time, tests were repeated until 72 h after umbilical cord collection. Total white blood cell count decreased in most cases, whereas absolute number of IMI-HPC tended to increase in time. In conclusion, if HPC measurements in the IMI channel are used to monitor circulating stem cells during mobilization, one has to be aware of a very low correlation between these results and those of other methods such as CD34(+) analysis and colony growth. False-negative results do occur, but if events are seen in the IMI channel, this simple monitoring technique is useful to predict the presence of circulating stem cells.     

The subcellular distribution of the nonspecific lipid transfer protein (sterol carrier protein 2) in rat liver and adrenal gland

The distribution of the nonspecific lipid transfer protein (i.e., sterol carrier protein 2) over the various subcellular fractions from rat liver and adrenal gland was determined by enzyme immunoassay and immunoblotting. This distribution is very different in each of these two tissues. In liver, 66% of the transfer protein is present in the membrane-free cytosol as compared to 19% in the adrenal gland. In the latter tissue, the transfer protein is mainly found in the lysosomal/peroxisomal and the microsomal fraction at a level of 1093 and 582 ng per mg total protein, respectively (i.e., 17% and 35% of the total), and to a lesser extent in the mitochondrial fraction (11% of the total). Of all the membrane fractions isolated, the microsomal fraction from the liver and the mitochondrial fraction from the adrenal gland have the lowest levels of the transfer protein (i.e., 168 ng and 126 ng per mg total protein, respectively). These low levels correlate poorly with the active role proposed for this transfer protein in the conversion of cholesterol into bile acids and steroid hormones in these fractions. Using immunoblotting, it was demonstrated that in addition to the transfer protein (14 kDa) a cross-reactive 58 kD protein was present in the supernatant and the membrane fractions of both tissues. Cytochemical visualization in adrenal tissue with specific antibodies against the nonspecific lipid transfer protein showed that immunoreactive protein(s) were present mainly in the peroxisome-like structures.     

Verification of ELISA results by immunomagnetic isolation of antigens from extracts and analysis with SDS-PAGE and Western blotting, demonstrated for Erwinia spp. in potatoes

Isolation of antigens on immunomagnetic beads and subsequent analysis with SDS-PAGE and Western blotting (immunomagnetic isolation-Western blotting (IMI-WB)) was used to verify positive ELISA results for Erwinia chrysanthemi and Erw. carotovora subsp. atroseptica in potato peel extracts. Direct analysis of highly contaminated extracts by Western blotting without previous immuno-isolation resulted in background reactions, whereas immunomagnetic isolation resulted in distinct bands of specific antigens. Target cells as well as antigenic cell products were captured in IMI-WB. Band patterns on IMI-WB of cell-free culture filtrates and cell suspensions were highly similar, but the removal of cells lowered the detection level by 10- to 100-fold. Threshold levels of IMI-WB were generally comparable with those of ELISA.
No differences in threshold levels and band patterns were found between a direct format and an indirect format of immuno-isolation.
In IMI-WB, blotting patterns differed between Erw. chrysanthemi and Erw. carotovora subsp. atroseptica. The patterns were identical for 15 Erw. chrysanthemi strains, isolated from potato peel extracts in The Netherlands. However, one of 15 strains of Erw. carotovora subsp. atroseptica from potato peel extracts in The Netherlands gave an aberrant pattern. Target bacteria could be easily distinguished from those of cross-reacting strains on the basis of band patterns.
Potato peel extracts naturally contaminated with Erw. chrysanthemi gave IMI-WB patterns that were similar to pure cultures of the homologous strains.     

Polymerase chain reaction for verification of fluorescent colonies of Erwinia chrysanthemi and Pseudomonas putida WCS358 in immunofluorescence colony staining

The potential of polymerase chain reaction (PCR) for verifying the identity of colonies stained by the immunofluorescence colony-staining (IFC) procedure was investigated. Using primers directed against conserved sequences of the pectate lyase-genes coding for isozymes PLa, PLd and PLe of Erwinia chrysanthemi , the authors confirmed the identity of 96% of 20 fluorescent target colonies, punched from IFC-stained samples with pure cultures. In pour plates with mixtures of Erw. chrysanthemi and non-target colonies from potato peel extracts, the identity of 90% of 113 target colonies was confirmed.
Using primers directed against sequences of the ferric-pseudobactin receptor gene pupA of Pseudomonas putida WCS358, the identity of 96% of 22 target colonies was confirmed in IFC-stained samples with pure cultures. In pour plates with mixtures of Ps. putida WCS358 and non-target bacteria from compost extracts, the identity of 59% of 108 fluorescent colonies was confirmed by PCR. It was shown that components from non-target bacteria lowered the threshold level of PCR for Ps. putida WCS358     

Detection of Clavibacter michiganensis subsp. sepedonicus by AmpliDet RNA,a new technology based on real time monitoring of NASBA amplicons with a molecular beacon

AIMS: To develop a procedure for direct detection of viable cells of Clavibacter michiganensis subsp. sepedonicus (Cms), the causal organism of bacterial ring rot in potato, based on AmpliDet RNA, in which amplicons generated by nucleic acid sequence based amplification (NASBA) are monitored in real time with a molecular beacon. METHODS AND RESULTS: Five methods were evaluated and fine-tuned for extraction of RNA from Cms. The most efficient non-commercial RNA extraction method included an enzymatic breakdown of the cell wall followed by a phenol extraction. AmpliDet RNA enabled detection of 10,000 molecules of purified rRNA per reaction and 100 cfu of Cms per reaction in more complex samples. Two primer pairs were tested with DNA and RNA purified from Cms. One primer pair was able to distinguish live from dead cells. CONCLUSIONS: An AmpliDet RNA was developed which enabled fast and specific detection of viable cells of Cms in complex substrates at a detection limit of 100 cfu per reaction. SIGNIFICANCE AND IMPACT OF THE STUDY: This novel AmpliDet RNA is carried out in sealed tubes, thus reducing the risk of carry-over contamination. The method will be particularly suitable for studies on the epidemiology of Cms in which viable cells should be exclusively detected.     

Multi-laboratory evaluation of procedures for reducing the volume of cord blood: influence on cell recoveries

BACKGROUND: Various procedures can be used to isolate stem and progenitor cells from cord blood. This study evaluated the hydroxyethyl starch sedimentation (HES) with two centrifugation steps, and the top and bottom (T&B) isolation of buffy coat following a single centrifugation, and two filter systems for processing cord blood, one developed by Asahi Kasei Medical (filter A) and the second by Terumo (filter B). METHODS: Each of seven laboratories was randomly assigned the evaluation of either the HES or T&B method and one of the filter methods (n=8 cord blood units, per laboratory, for each method). The leukocyte-containing fraction with the stem/progenitor cells was recovered from the filters by reverse flushing. Utilizing the routine traditional processing and testing procedures of each laboratory, in vitro parameters were determined, with samples obtained after collection, after processing and after freezing/thawing. The results were expressed as the percentage recovery of viable cells in processed vs. collected samples (performance 1; PF1) and in thawed vs. processed samples (performance 2; PF2). The composite results obtained by the seven laboratories were summarized. RESULTS: The median PF1 percentage recovery of total nucleated cells (TNC) was comparable with both traditional methods (HES 79%, T&B 86%) and statistically reduced with both filtration procedures (filter A 58%, filter B 61%). Mononuclear cell (MNC) PF1 recovery was highest statistically with the T&B method (91%) and reduced on using filter A (77%) and filter B (70%) and the HES method (72%). CD34+ cell recovery was judged to be essentially comparable with the four methods, although the range of unit recoveries differed. The percentage recovery of TNC and MNC in PF1 was influenced by the volume of the collected cord blood, especially with use of the filtration procedures. This correlated with TNC content. A greater percentage of red cells and platelets was removed during processing with both filter methods. The time to process cord blood preparations with filter A was significantly shorter than the other methods. Processing with the HES method took the longest time. The recoveries for TNC, MNC and CD34+ cells in PF2 did not appear to be influenced by the specific processing procedure. DISCUSSION: These data indicate that filters that capture stem and progenitor cells may be an appropriate methodology for processing cord blood collected for banking.     

Fate of Clavibacter michiganensis ssp. sepedonicus, the Causal Organism of Bacterial Ring Rot of Potato, in Weeds and Field Crops

Crops and weeds were tested for their ability to host Clavibacter michiganensis ssp. sepedonicus (Cms), the causal agent of bacterial ring rot in potato. Ten crops grown in rotation with potato in Europe, namely maize, wheat, barley, oat, bush bean, broad bean, rape, pea and onion and five cultivars of sugar beet were tested by stem and root inoculation. About 6–8 weeks after inoculation, Cms could be detected in most crops except onion and sugar beet, in larger numbers in stems (10⁵–10⁶ cells/g of tissue) than in roots (≤10³ cells/g of tissue) in immunofluorescence cell‐staining (IF). Cms was successfully re‐isolated only from IF‐positive stem samples of maize, bush bean, broad bean, rape and pea, but not from roots. Twelve solanaceous weeds and 13 other weeds, most commonly found in potato fields in Europe, were tested in IF as hosts of Cms by stem and root inoculations. Only in Solanum rostratum, a weed present in northern America, Cms persisted in high numbers (10⁸ cells/g tissue) in stems and leaves, where it caused symptoms. In the other solanaceous weeds, Cms persisted at low numbers (approximately 10⁵ cells/g of tissue) in stems but less so in roots. The bacteria could be frequently re‐isolated from stem but not from root tissues. In 2 consecutive years, plants from 14 different weed species were collected from Cms‐contaminated potato field plots and tested for the presence of Cms by dilution plating or immunofluorescence colony‐staining (IFC), and by AmpliDet RNA, a nucleic acid‐based amplification method. Cms was detected in roots but not in stems of Elymus repens plants growing through rotten potato tubers, and in some Viola arvensis and Stellaria media plants, where they were detected both in stems and roots, but more frequently by AmpliDet RNA than by IFC.     

Factors Affecting Survival of Clavibacter michiganensis subsp. sepedonicus in Water

The survival of Clavibacter michiganensis subsp. sepedonicus (Cms), the causal organism of bacterial ring rot in potato, was studied in water, to assess the risks for dissemination of Cms via surface water and infection of potato crops by irrigation. Cms was able to survive for a maximum period of 7 days in non‐sterile surface water at 10°C, a period during which Cms can be transported over long distances, but will also be strongly diluted. It is concluded that contamination of surface water with Cms can pose a threat on potato production only if aquatic host plants can multiply Cms in high densities. Survival of a fluidal and non‐mucoid strain was also studied in sterile ditch water and simulated ‘drainage water’, in sterile MilliQ water, in tap water, in physiological salt and in artificial xylem fluid. In addition, the influence of temperature and low oxygen conditions on persistence of Cms in some of these diluents was studied. A maximum survival period of 35 days was found for Cms in sterile tap water at 20°C, independent of the strain used. In the other diluents survival periods ranged between 0 and 21 days. Relatively poor survival was found in MilliQ water and artificial xylem fluid. Low temperatures of 4°C do not favour survival as it does in soil. Oxygen depletion affected survival detrimentally. Survival periods determined by agar dilution plating and a direct viable counting method, based on the use of indicators for esterase activity and membrane integrity were similar. Therefore, it was concluded that under the experimental conditions studied, Cms did not form cells in a viable but non‐culturable state.