A gel electrophoretic technique for the rapid and sensitive detection of viroids and virusoids is described. Starting from plant material, a typical analysis requires less than 5 hours. Viroid concentrations as low as 800 pg/g tissue can be detected unambiguously without the use of radioactivity, organic solvents, or highly specialized laboratory equipment. The sensitivity may be further increased by introducing additional purification steps. The technique is an essential improvement of the previously published bidirectional gel electrophoretic analysis (Schumacheret al.1983, Anal. Biochem. 135, 288–295). In the new procedure gel electrophoresis is first carried out under native conditions. Before the viroid (or virusoid) bands will leave the gel, conditions are changed to provide denaturing conditions which are achieved by increasing the temperature and changing the buffer. After changing the polarity of the electric field all nucleic acids in the gel “return” in that they now migrate towards their original starting point. Under the denaturing conditions in the second electrophoresis viroids (or virusoids) unfold into the conformation of a circle without in tramolecular base pairs, which structure is unique among the nucleic acids in the gel. The denatured circular viroids migrate in the gel much slower than all other nucleic acids of comparable molecular weight and, therefore stay well separated behind the edge of the other nucleic acids. Thus, viroids can easily be detected on the stained gel as a discrete band.

Internal stress to chromium is only relevant in occupational medicine if it is due to the handling of hexavalent chromium. Cr(VI) ions, after uptake by inhalation or percutaneously are carried in the blood plasma and penetrate--depending on the concentration--into the erythrocytes. Due to the intracellular reduction to Cr(III) and the concurrent intracellular protein binding, the erythrocytes represent an easily accessible target organ for quantitative chromium determination after occupational exposure to Cr(VI) compounds. The results of an earlier experimental study indicate that human plasma too is capable of spontaneous reduction of Cr(VI) ions of up to 2 ppm to Cr(III). This plasma reduction capacity (PRC) can be increased and accelerated considerably by adding ascorbic acid (AA). These findings were supported in this investigation by proving a decreased binding of Cr(VI) inside the erythrocytes under the effect of AA. This leads to the assumption that only those Cr(VI) concentrations can penetrate the membrane of the erythrocytes and enter the cell which either come into contact with the membrane during the reduction process or exceed this limit concentration of 2 ppm. Only in these two instances can corresponding chromium findings be analyzed in isolated and washed erythrocytes. These results are compared with those obtained by conventional methods, such as Cr determination in the blood and/or urine. Our findings indicate that a single determination of chromium concentration in the erythrocytes will permit the monitoring of critical cases of Cr(VI) exposure. This is a new type of biological monitoring in the sense of a condensed longitudinal study, in order to find out whether threshold concentrations have been respected over a given period.

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SummaryA test procedure for PSTVd is described based on immobilisation of plant sap on filter paper, by dotting or tissue printing followed by RT-PCR. Tests were carried out using primarily and secondarily infected potato plants, primarily infected in vitro plants, and potato tubers. Print PCR was shown to be suitable for testing large samples of potato plants whereas dot PCR is recommended for in vitro plantlets and tuber tissue. Bulking one infected plant to 4 or 9 healthy plants gave reliable results with secondarily infected potato plants, but sometimes the test failed to detect PSTVd in primarily infected in vitro plants. Dotted and printed paper squares could be stored at 4 ~ for at least 2 weeks in Triton X-100 solution or under dry conditions. Storing at room temperature can lead to unreliable results.

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