Development of a universal endogenous qPCR control for eukaryotic DNA samples

Abstract

Background: Phytoplasma 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. Results: We 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. Conclusions: Quality 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.

Surveying Potential Vectors of Apple Proliferation Phytoplasma: Faunistic Analysis and Infection Status of Selected Auchenorrhyncha Species

“…The phytoplasma titer was quantified based on the four-point plasmid standard curve analyzed in parallel with the samples in each PCR run. Due to size and morphological differences between the tested insect species, the DNA yield and the potential presence of PCR inhibitors might vary strongly in DNA extraction samples from different insect species [ 59 ]. To verify that the diagnostic detection of ’ Ca .…”

“…Each species was initially spike-tested using SYBR-based quantitative PCR. However, SYBR analysis is error-prone if high concentrations of double-stranded DNA are present in the sample background [ 59 ]. In case no reliable phytoplasma detection was achieved in the SYBR spike-controls, the analysis was repeated using the 16S specific primers, the hydrolysis probe, and the respective plasmid, as described above.…”

Symptomatology, (Co)occurrence and Differential Diagnostic PCR Identification of ‘Ca. Phytoplasma solani’ and ‘Ca. Phytoplasma convolvuli’ in Field Bindweed

“…P. solani'-and of 'Ca. P. convolvuli'-specific primer pairs Tuf-Sol-Fq/Rq and Tuf-Con-Fq/Rq and the primers for the 28S rDNA gene [48] amplifying Convolvulus arvensis as endogenous control.…”

“…To validate the DNA preparation and DNA integrity separate SYBR Green-based realtime PCR amplifications targeting eukaryotic 28S rDNA were performed on the plant DNA gene of all C. arvensis samples as an endogenous control. Primers UNI28S-fwd/rev [48] were used under the same amplification conditions described above, except that primer concentrations were 250 nM each, as recommended [48]. The potential for multiplex amplification of all three targets in a single reaction using 'Ca.…”

Analytical approaches of meat authentication in food

“…Some instances are mtDNA 16S rRNA conserved sequence which has been aligned to amplify both mammalian and avian species (Sawyer et al ., 2003), nDNA myostatin gene which serves as a control in amplifying mammalian and poultry species (Laube et al ., 2007) and nDNA 18S rRNA gene which can amplify conserved region among eukaryotes species (Rojas et al ., 2010). However, nDNA is commonly used as an endogenous or internal control in mtDNA‐based PCR assay (Martín et al ., 2009; Amaral et al ., 2017; Mittelberger et al ., 2020) as the sequences are more conserved among a group of species.…”