Direct detection of potato leafroll virus in potato tubers by immunocapture and the isothermal nucleic acid amplification method NASBA

NASBA, an isothermal amplification method for nucleic acids, was applied to the detection of RNA of potato leafroll virus (PLRV) in a single enzymatic reaction at 41 °C. A set of primers was selected from the coat protein open reading frame sequence of PLRV to allow amplification of viral RNA. The NASBA reaction products were visualized after electrophoresis by ethidium bromide or acridine orange staining. The specificity of the amplification products was validated by Northern blot analysis with a PLRV-specific 32P-labelled oligonucleotide probe. The procedure was coupled to immunocapture of PLRV virions from tuber extracts by immobilized antibodies in microtubes. It was possible to discriminate readily by this method between uninfected and primarily PLRV-infected potato tubers. NASBA is suitable for the direct detection of PLRV in potato tubers from primarily infected plants, offering the potential to considerably simplify the inspection of seed-potatoes for virus infection

Direct detection of potato leafroll virus in potato tubers by immunocapture and the isothermal nucleic acid amplification method NASBA

NASBA, an isothermal amplification method for nucleic acids, was applied to the detection of RNA of potato leafroll virus (PLRV) in a single enzymatic reaction at 41 °C. A set of primers was selected from the coat protein open reading frame sequence of PLRV to allow amplification of viral RNA. The NASBA reaction products were visualized after electrophoresis by ethidium bromide or acridine orange staining. The specificity of the amplification products was validated by Northern blot analysis with a PLRV-specific 32P-labelled oligonucleotide probe. The procedure was coupled to immunocapture of PLRV virions from tuber extracts by immobilized antibodies in microtubes. It was possible to discriminate readily by this method between uninfected and primarily PLRV-infected potato tubers. NASBA is suitable for the direct detection of PLRV in potato tubers from primarily infected plants, offering the potential to considerably simplify the inspection of seed-potatoes for virus infection

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