Development of multiplex RT-PCR assays containing an internal amplification control for the detection of dicistro-, iflaviruses and CBPV in honey bees. Part 1 - assays design and optimization

Dagmara Zdańska ¹, Artur Rzeżutka ², Krystyna Pohorecka ³

⁰Department of Parasitology and Invasive Diseases, Bee Diseases and Aquatic Animal Diseases, National Veterinary Research Institute, Al. Partyzantów 57, Puławy, 24-100, Poland. dagmara.zdanska@piwet.pulawy.pl.

Veterinary Research Communications +

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January 15, 2026

View abstract on PubMed

Summary

New multiplex RT-PCR assays enable simultaneous detection of six common honey bee viruses. These validated methods improve diagnostics for asymptomatic infections, aiding in the protection of vital honey bee populations.

Area Of Science

Veterinary Virology
Apiculture Science
Molecular Diagnostics

Background

Honey bee colonies face significant mortality due to viral infections.
Asymptomatic viral infections in bees necessitate sensitive diagnostic tools.
Current conventional RT-PCR assays lack documented validation for honey bee virus detection.

Purpose Of The Study

To develop and validate two in-house multiplex RT-PCR (mRT-PCR) assays.
To detect multiple honey bee viruses including CBPV, iflaviruses (DWV-A, SBV), and dicistroviruses (ABPV, IAPV, BQCV).
To incorporate an internal amplification control (IAC) for enhanced assay reliability.

Main Methods

Development of two mRT-PCR assays: Ifla-CBPV and Dicistro.
Primer sequence refinement to include global and local honey bee virus genetic variations.
Optimization of PCR reaction components and thermal cycling conditions.
Incorporation of an Internal Amplification Control (IAC) into both assays.

Main Results

Successfully developed two IAC-controlled mRT-PCR assays.
Assays enable simultaneous detection of six common honey bee virus species.
Validated assays provide reliable detection of mixed viral infections.
IAC incorporation monitors assay performance, preventing false negatives.

Conclusions

The developed IAC-controlled mRT-PCR assays offer a validated and sensitive method for detecting multiple honey bee viruses.
These assays are crucial for monitoring asymptomatic infections and understanding global bee virus epidemiology.
The validated diagnostic tools support efforts to protect honey bee health and colony survival.

Keywords:

Detection Honey bee Internal amplification control Multiplex RT-PCR Viruses