Amplification of RNA by NASBA allows direct detection of viable cells of <i>Ralstonia solanacearum</i> in potato

Bentsink, Leónie; Leone, G.; Beckhoven, J.R.C.M. van; Schijndel, Harm B. van; Gemen, Bob van; Wolf, J.M. van der

doi:10.1046/j.1365-2672.2002.01725.x

Cited by 34 publications

(25 citation statements)

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“…Serological techniques rely on the detection of bacterial components using antibodies [18], while molecular techniques are based on the detection of bacterial DNA [19], [20], or RNA of viable bacterial cells [21]. To control the spread of R. solanacearum , a rapid and more user-friendly test is needed that can be easily deployed on-site.…”

Section: Introductionmentioning

confidence: 99%

Loop-Mediated Isothermal Amplification of Specific Endoglucanase Gene Sequence for Detection of the Bacterial Wilt Pathogen Ralstonia solanacearum

et al. 2014

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The increased globalization of crops production and processing industries also promotes the side-effects of more rapid and efficient spread of plant pathogens. To prevent the associated economic losses, and particularly those related to bacterial diseases where their management relies on removal of the infected material from production, simple, easy-to-perform, rapid and cost-effective tests are needed. Loop-mediated isothermal amplification (LAMP) assays that target 16S rRNA, fliC and egl genes were compared and evaluated as on-site applications. The assay with the best performance was that targeted to the egl gene, which shows high analytical specificity for diverse strains of the betaproteobacterium Ralstonia solanacearum, including its non-European and non-race 3 biovar 2 strains. The additional melting curve analysis provides confirmation of the test results. According to our extensive assessment, the egl LAMP assay requires minimum sample preparation (a few minutes of boiling) for the identification of pure cultures and ooze from symptomatic material, and it can also be used in a high-throughput format in the laboratory. This provides sensitive and reliable detection of R. solanacearum strains of different phylotypes.

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Section: Introductionmentioning

confidence: 99%

Loop-Mediated Isothermal Amplification of Specific Endoglucanase Gene Sequence for Detection of the Bacterial Wilt Pathogen Ralstonia solanacearum

et al. 2014

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“…99 The utility and application of NASBA for direct detection and quantification of viable Ralstonia solanacearum cells has also been reported with an LOD of 10 4 copies per reaction. 100 …”

Section: Direct Amplification Studies Using Isothermal or Pcr Polymermentioning

confidence: 99%

Implications of direct amplification for measuring antimicrobial resistance using point-of-care devices

et al. 2017

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Antimicrobial resistance (AMR) is recognized as a global threat to human health. Rapid detection and characterization of AMR is a critical component of most antibiotic stewardship programs. Methods based on amplification of nucleic acids for detection of AMR are generally faster than culture-based approaches but they still require several hours to more than a day due to the need for transporting the sample to a centralized laboratory, processing of sample, and sometimes DNA purification and concentration. Nucleic acids-based point-of-care (POC) devices are capable of rapidly diagnosing antibiotic-resistant infections which may help in making timely and correct treatment decisions. However, for most POC platforms, sample processing for nucleic acids extraction and purification is also generally required prior to amplification. Direct amplification, an emerging possibility for a number of polymerases, has the potential to eliminate these steps without significantly impacting diagnostic performance. This review summarizes direct amplification methods and their implication for rapid measurement of AMR. Future research directions that may further strengthen the possibility of integrating direct amplification methods with POC devices are also summarized.

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“…This corresponds with less than one bacterial cell, assuming that a metabolically active cell contains ca. 10 5 copies of RNA (Bentsink et al 2002).…”

Section: Nucleic Acid Sequence-based Amplification Assaymentioning

confidence: 99%

Detection of Bacterial and Phytoplasmal Pathogens

Narayanasamy¹

2010

Microbial Plant Pathogens-Detection and Disease Diagnosis:

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Bacterial pathogens classified as prokaryotes are much simpler in structure and smaller in size and they have less complex structural features compared to fungal plant pathogens. As the morphological characteristics of bacterial cells are less variable, biological, biochemical, physiological, immunological and genomic characteristics have to be determined for reliable identification and meaningful classification of bacterial pathogens. Detection and identification of bacterial plant pathogens present in whole plants and in propagative plant materials have been possible by employing isolation on cultural media and metabolic fingerprinting methods. But they are labor-intensive and require long time. Results often are inconclusive. Isozyme analysis, direct colony thin layer chromatography and gel electrophoresis techniques have been successfully applied for the detection of some bacterial pathogens. Immunoassays and nucleic acid-based assays have become widely accepted techniques, providing more sensitive and specific detection and quantification of bacterial pathogens affecting a wide range of host plant species. However, the major limitation of the molecular techniques is their inability to discriminate living cells from the dead ones. Attempts have been made to overcome this limiting factor by using DNA binding dyes and estimating pathogen RNAs as an indicator of cell viability. The diagnostic methods have both merits and demerits and hence, appropriate method has to be selected based on cost-effectiveness and possibility of obtaining reliable results rapidly to suit the requirements of the investigation concerned.Phytoplasmas are cell wall-less, nonculturable bacteria belonging to Mollicutes. Lack of cultural characteristics has made obligatory to study the morphological characteristics of phytoplasmas present in the phloem cells of infected plant hosts by observing under electron microscopes. As most of the phytoplasmas look alike in the ultrathin sections, the morphological characters have no diagnostic value. Histochemical methods using DNA binding dyes have been employed to localize the phytoplasmas in the host cells. Application of immunoassays and nucleic acidbased techniques has been effective in providing information for the identification and differentiation of phytoplasams. Polyclonal and monoclonal antibodies have been generated for detection of phytoplasmas infecting several plant species. Universal and species-specific primers and probes have been designed based on

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

Cited by 34 publications

References 30 publications

Loop-Mediated Isothermal Amplification of Specific Endoglucanase Gene Sequence for Detection of the Bacterial Wilt Pathogen Ralstonia solanacearum

Loop-Mediated Isothermal Amplification of Specific Endoglucanase Gene Sequence for Detection of the Bacterial Wilt Pathogen Ralstonia solanacearum

Implications of direct amplification for measuring antimicrobial resistance using point-of-care devices

Detection of Bacterial and Phytoplasmal Pathogens

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