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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, PCRamplification and dilution plating on the semiselective medium SMSA. A positive correlation was found between NASBA and dilution plating detecting culturable cells, whereas PCRamplification 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 10 4 cfu per ml of extract, equivalent to 100 cfu per reaction. With purified RNA a detection level of 10 4 rRNA molecules was found. This corresponds with less than one bacterial cell, assuming that a metabolically active cell contains ca 10 5 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.

We have established that a recombinant von Willebrand Factor (vWF) mutant (vWFdelpro) that lacks the propolypeptide, in contrast with mature wild-type vWF, with which it is identical in terms of primary amino acid sequence, is not able to form a complex with Factor VIII. Wild-type vWF (flvWF) and vWFdelpro were expressed in AtT-20 cells. Under the culture conditions employed, completely processed multimerized flvWF and dimeric vWFdelpro were secreted into the medium. FlvWF and vWFdelpro were compared for their Factor VIII-binding properties in two distinct assay systems. In a direct binding assay, purified human Factor VIII was shown to bind to flvWF that had been immobilized on the surface of microtitre wells by using an anti-vWF monoclonal antibody. In contrast, Factor VIII did not bind to immobilized vWFdelpro. In a competition assay, fluid-phase flvWF appeared to inhibit efficiently the binding of Factor VIII to immobilized vWF isolated from plasma, whereas vWFdelpro did not influence Factor VIII binding. From these observations, it is argued that the pro-polypeptide serves an essential role in the post-translational processes that lead to the expression of a functional Factor VIII-binding site on the mature vWF subunit.

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