POSTERS: Pathogen detection, quantification and diagnosis
An RNA Extraction Method for Improved Molecular Diagnostics of Plum Pox Virus in PPQ-CPHST Beltsville Laboratory
Gang Wei - USDA-APHIS-PPQ-S&T-CPHST. Vessela Mavrodieva- USDA-APHIS-PPQ S&T CPHST, Benjamin Adducci- USDA-APHIS-PPQ-S&T-CPHST
Plum pox virus (PPV) is a potyvirus that infects all known commercial, ornamental, wild Prunus spp. and some weeds. PPV was first reported in the USA in 1999 and has been under federal quarantine and eradication ever since. For federal confirmatory diagnostics, any PPV-suspect sample identified by screening laboratories is forwarded to the CPHST Beltsville Lab. As a part of the confirmatory protocol, Immunocapture (IC) of PPV particles was performed prior to RT-PCR analysis. IC allows for using the same ground sample prepared for DAS ELISA, thus overcoming the challenge of PPV’s uneven distribution in the sample. Improvements were identified for the current system: a need of a plant internal control for improved reliability of RT-PCR and a need to overcome defined specificity of antibodies that may not capture all variants of the virus. We have developed a total RNA extraction method starting from the same DAS ELISA sample suspension. A real-time RT-PCR assay was used to validate the RNA extraction method. We found that the total RNA extraction method is more sensitive than IC, as it produced lower Ct values. When using the same amount of starting suspension (100 µL) as IC, total RNA extraction produced lower Cts by an average of 1.92. When the suspension volume was increased to 300 µL, the RNA extraction method produced an average of 3.94 Cts lower than IC. Additionally, the total RNA extraction method was found to be faster and easier to perform than IC.