Simultaneous detection of three pome fruit tree viruses by one-step multiplex quantitative RT-PCR

Area of Science:

• Plant Pathology
• Molecular Biology
• Virology

Background:

• Apple mosaic virus (ApMV), Apple stem pitting virus (ASPV), and Apple stem grooving virus (ASGV) are significant pathogens affecting pome trees.
• Accurate and efficient detection methods are essential for managing these viral diseases and for certification programs.

Purpose of the Study:

• To develop a one-step multiplex real-time reverse transcription polymerase chain reaction (RT-qPCR) assay for simultaneous detection of ApMV, ASPV, and ASGV.
• To evaluate the sensitivity and efficiency of the developed RT-qPCR assay compared to conventional methods.

Main Methods:

• Development of a multiplex real-time RT-qPCR assay using TaqMan probes for simultaneous detection of three apple viruses.
• RNA extraction from pome trees using the CTAB method.
• Sensitivity assessment using serial dilutions of in vitro synthesized viral transcripts and field samples.

Main Results:

• The developed RT-qPCR assay successfully detected ApMV, ASPV, and ASGV simultaneously.
• The assay demonstrated high sensitivity, being at least 100 times more sensitive than conventional single RT-PCRs.
• Validation with field samples identified nine triple and 11 double infections, with detection possible in composite samples at a 1:150 ratio.

Conclusions:

• The one-step multiplex real-time RT-qPCR assay provides a rapid, sensitive, and efficient tool for simultaneous detection of ApMV, ASPV, and ASGV.
• This assay is suitable for large-scale sample analysis in pome tree certification programs and virus surveys.
• The method offers a significant advancement over traditional virus detection techniques.