ArticlePlant methods2020
Development of a universal endogenous qPCR control for eukaryotic DNA samples.
Article in Plant methods, 2020. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 9 papers.
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Who cites it
9 citing papers in PubMed, 28 citations in OpenAlex.
- Molecular monitoring and multiplex qPCR-based detection ofFrontiers in insect science · 2026Article
- Hyperspectral differentiation of three grapevine yellows diseases and symptomatically similar stresses.Frontiers in plant science · 2026Article
- Sunflower as a 'Frontiers in plant science · 2026Article
- 'Candidatus Phytoplasma mali' SAP11-Like protein modulates expression of genes involved in energy production, photosynthesis, and defense in Nicotiana occidentalis leaves.BMC plant biology · 2024Article
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- Rapid and Accurate Detection ofPathogens (Basel, Switzerland) · 2022Article
- The Use of qPCR to Detect Cryphonectria parasitica in Plants.Methods in molecular biology (Clifton, N.J.) · 2022Article
- Symptomatology, (Co)occurrence and Differential Diagnostic PCR Identification of 'Pathogens (Basel, Switzerland) · 2021Article
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Authors and funding
9 authors at 1 institution in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
backgroundPhytoplasma are obligate intracellular plant-pathogenic bacteria that infect a broad range of plant species and are transmitted by different insect species. Quantitative real-time PCR (qPCR) is one of the most commonly used techniques for pathogen detection, especially for pathogens that cannot be cultivated outside their host like phytoplasma. PCR analysis requires the purification of total DNA from the sample and subsequent amplification of pathogen DNA with specific primers. The purified DNA contains mainly host DNA and only a marginal proportion is of phytoplasmal origin. Therefore, detection of phytoplasma DNA in a host DNA background must be sensitive, specific and reliable and is highly dependent on the quality and concentration of the purified DNA. DNA quality and concentration and the presence of PCR-inhibitors therefore have a direct impact on pathogen detection. Thus, it is indispensable for PCR-based diagnostic tests to validate the DNA preparation and DNA integrity before interpreting diagnostic results, especially in case that no pathogen DNA is detected. The use of an internal control allows to evaluate DNA integrity and the detection of PCR-inhibiting substances. Internal controls are generally host-specific or limited to a defined group of related species. A control suitable for the broad range of phytoplasma hosts comprising different insect and plant species is still missing.
resultsWe developed a primer and probe combination that allows amplification of a conserved stretch of the eukaryotic 28S rDNA gene. The developed endogenous qPCR control serves as a DNA quality control and allows the analysis of different eukaryotic host species, including plants, insects, fish, fungi, mammals and human with a single primer/probe set in single- or multiplex assays.
conclusionsQuality and performance control is indispensable for pathogen detection by qPCR. Several plant pathogens are transmitted by insects and have a broad range of host species. The newly developed endogenous control can be used with all so far tested eukaryotic species and since multiplexing is possible, the described primer and probe set can be easily combined with other PCR-based pathogen detection systems.
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