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.
Highlights
Phytoplasma are obligate intracellular plant-pathogenic bacteria that infect a broad range of plant species and are transmitted by different insect species
This specific exogenous DNA must be detected by Polymerase chain reaction (PCR), as an internal amplification control in sufficient amounts after extraction to ensure that DNA was successfully purified from the sample
The use of exogenous controls allows to analyze the presence of potential PCR inhibitors in the sample
Summary
Phytoplasma are obligate intracellular plant-pathogenic bacteria that infect a broad range of plant species and are transmitted by different insect species. To analyze the unwanted loss of DNA during a purification procedure exogenous DNA of known sequence can be added to a sample prior to DNA extraction [6] This specific exogenous DNA must be detected by PCR, as an internal amplification control in sufficient amounts after extraction to ensure that DNA was successfully purified from the sample. The use of exogenous controls allows to analyze the presence of potential PCR inhibitors in the sample This method is based on the addition of an external DNA fragment and does not deliver information about the integrity of the target DNA that is of real interest [7]
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