Development of a universal endogenous qPCR control for eukaryotic DNA samples

Area of Science:

• Molecular Biology
• Plant Pathology
• Entomology

Background:

• Phytoplasmas are plant-pathogenic bacteria with broad host ranges, transmitted by insects.
• Quantitative real-time PCR (qPCR) is crucial for detecting non-culturable phytoplasmas, but requires high-quality DNA.
• Existing internal controls are host-specific, lacking a universal solution for diverse plant and insect hosts.

Purpose of the Study:

• To develop a universal endogenous control for qPCR to assess DNA quality and detect inhibitors.
• To enable reliable phytoplasma detection across various eukaryotic hosts.

Main Methods:

• Developed a primer and probe set targeting a conserved region of the eukaryotic 28S rDNA gene.
• Validated the primer/probe set for amplification in diverse eukaryotic species (plants, insects, fish, fungi, mammals, human).
• Demonstrated utility in single- and multiplex qPCR assays.

Main Results:

• A novel primer and probe combination successfully amplifies a conserved eukaryotic 28S rDNA sequence.
• The developed endogenous control functions as a DNA quality control across a wide range of eukaryotic species.
• The control is effective in single- or multiplex assays, compatible with various host types.

Conclusions:

• Endogenous controls are essential for validating qPCR-based pathogen detection.
• The new universal control is suitable for diverse eukaryotic hosts, including plants and insects.
• This tool enhances the reliability of phytoplasma detection and can be integrated with other PCR-based diagnostic systems.