The Impact of the Product Generation Life Cycle on Knowledge Valorization at the Public Private Research Partnership, the Centre for BioSystems Genomics

The present paper aims to address the impact of the product generation life cycle (PGLC) on knowledgevalorization in public private research partnerships (PPRPs). Data were collected from participants in theCentre for BioSystems Genomics (CBSG), a Dutch PPRP program in the plant breeding sector. In total, 15commercial partners participated in the study, 7 with a relatively short PGLC of 5 to 6 years, active in thetomato sector, and 8 potato partners, having a very long PGLC of up to 25 years. The results show a clearrelationship between CBSG’s valorization support activities and the level of knowledge utilization by theparticipants, although the preferred type of support activities differs between the potato and tomatocompanies. Firms with a long PGLC, having a higher complicacy of the R&D process, require more basicresearch support and extra communication tools that help to bridge the gaps caused by the long durationof the development process. Companies with short PGLCs, being challenged to keep development timeof new products as short as possible in order not to miss out on market opportunities, value the PPRPmost for the networking possibilities and as provider of the latest technological developments

Development of a multiplex AmpliDet RNA for the simultaneous detection of Potato leafroll virus and Potato virus Y in potato tubers

A novel isothermal multiplex AmpliDet RNA system is described for the simultaneous amplification and detection of Potato leafroll virus (PLRV) and Potato virus Y (PVY) in seed potatoes. The risk of contamination by carry-over during diagnostic screening is eliminated by performing the reaction in a single closed tube. The viruses present in a sample are identified using differently coloured molecular beacons directed to a selected virus-specific sequence within the amplicon formed during amplification. With this system, as little as 10 fg of purified PLRV or PVY can be detected. The presence of both viruses in a sample is detected by the multiplex assay within a high range of virus concentrations. The reliability of the multiplex assay was compared with the enzyme-linked immunosorbent assay for detection of PLRV- or PVY-antigens in potato tubers. The multiplex assay detected clearly the viruses present originally in the potato tubers in all samples, demonstrating its potential for routine diagnostic work and high-throughput screening

Specific detection of Ralstonia solanacearum 16S rRNA sequences by AmpliDet RNA

The potential of AmpliDet RNA for specific detection of Ralstonia solanacearum in potato tuber samples and surface water was demonstrated. AmpliDet RNA is a procedure based on nucleic acid sequence based amplification (NASBA) of RNA sequences and homogeneous real time detection of NASBA amplicons with a molecular beacon. The procedure is carried out in sealed tubes, thus reducing the risks for carry-over contamination. AmpliDet RNA enabled reliable detection of specific 16S rRNA sequences of R. solanacearum in total RNA extracts from potato tuber samples in 90 min at a level of 10 cells per reaction, equivalent to ca. 104 cells ml-1 of sample. In surface water, AmpliDet RNA allowed detection of R. solanacearum at a level of 10 cfu ml-1, after concentrating bacteria from 200 ml of surface water into 1 ml of surface water by centrifugation. All strains of R. solanacearum and a strain of R. syzygii were positive in AmpliDet RNA, but not other (related) bacterial species. Ralstonia solanacearum (race 3, biovar 2) could be detected reliably in 18 naturally infected potato tuber samples containing varying concentrations of cells. Ninety-one negative tuber samples, from which no R. solanacearum was isolated, were tested in AmpliDet RNA, including 23 samples containing bacteria (cross-) reacting with antibodies against R. solanacearum in immunofluorescence (IF) cell-staining. Only one negative sample, containing high numbers of IF-positive cells, was positive in AmpliDet RN

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

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