Selection and Multiplexing of Reverse Transcription-Quantitative PCR Tests Targeting Relevant Honeybee Viral...

Area of Science:

• Molecular biology
• Apiculture
• Virology
• Diagnostic assay development

Background:

• Viral sequence variation challenges the reliability of molecular diagnostic methods for honeybee viruses.
• Accurate diagnosis of detrimental honeybee viruses is crucial for colony health and beekeeping sustainability.
• Existing diagnostic tools may lack inclusivity for diverse viral strains.

Purpose of the Study:

• To select and design inclusive probes for reverse transcription-quantitative PCR (RT-qPCR) assays targeting key honeybee viruses.
• To improve the reliability and inclusivity of molecular diagnostics for acute bee paralysis virus (ABPV), black queen cell virus (BQCV), chronic paralysis bee virus (CBPV), deformed wing virus variants A (DWVA) and B (DWVB), and sacbrood virus (SBV).
• To develop multiplex RT-qPCR assays capable of detecting multiple viral pathogens and variants simultaneously.

Main Methods:

• In silico re-evaluation of existing RT-qPCR methods for specificity and inclusivity.
• Modification of selected methods and design of new probes for enhanced inclusivity.
• Testing of developed assays in duplex RT-qPCR reactions, assessing limits of detection (LODs), multiplexing effects on sensitivity, and viral RNA quantification in bees and hive debris.

Main Results:

• Developed and validated inclusive RT-qPCR diagnostic assays for major honeybee viruses.
• The new assays demonstrated improved inclusivity, detecting a broader range of viral variants compared to previous methods.
• Successful simultaneous detection of two viral pathogens in a single RT-qPCR reaction was achieved, alongside assessment of LODs and quantification potential.

Conclusions:

• The study provides reliable and inclusive molecular diagnostic tools for honeybee viruses, enhancing disease surveillance.
• The developed multiplex RT-qPCR assays offer a more efficient and comprehensive approach to diagnosing viral infections in honeybees and hive debris.
• These advancements contribute to better understanding and management of honeybee viral diseases.