REAL TIME PCR METHOD FOR PPV DIAGNOSTIC ON PLUMS AND APRICOT IN THE REPUBLIC OF NORTH MACEDONIA

Abstract

Real-time PCR (Polymerase Chain Reaction) or qPCR is a method by which the amount of the PCR product can be determined in real-time, and is very useful for investigating gene expression. The main advantages of qPCR are that it provides fast and high-throughput detection and quantification of target DNA sequences in different matrices. The lower time of amplification is facilitated by the simultaneous amplification and visualization of newly formed DNA amplicons. The development and application of molecular methods for the detection of pathogens has significantly changed the diagnosis and control of plant diseases, various environmental samples, including hosts tissues, soil, water and air. With real time PCR method, it is possible not only to identify and detect the presence or absence of the target pathogen, but it is also possible to quantify the amount present in the sample allowing the quantitative assessment of the number of the pathogen in the sample. Detection and accurate identification of plant pathogens is one of the most important strategies for controlling plant diseases to initiate preventive or curative measures.

Plum pox virus (PPV), the agent of Sharka, is the most devastating virus infecting stone fruits. The PPV control is mainly based on prevention, and its quick and reliable detection is considered crucial in this strategy. In this study DAS-ELISA and real-time PCR were compared for evaluating their potentialities and limits for large scale surveys. Plum hosts (Prunus domestica) and apricot samples (Prunus armeniaca) from several different place are included for laboratory test analyzes, plant organs (phloem, buds, flowers, leaves and fruits) and parts of them, different periods of the year (spring, summer and winter period 2017/20), the presence or absence of symptoms were considered for comparison. Using DAS-ELISA tests, and use a universal set of antibodies (BIOREBA), has proved the presence of virus of Plum pox in all examined samples, especially from samples collected in summer, but also in checking virus status in winter and early spring season. Testing found high concentrations of viral antigens in plant samples (OD 2.912-2.752, for 30 min / 405 nm). Real time PCR show amplification plot for positive PPV samples on plums and apricot.