Development of a multiplex real-time quantitative reverse-transcription polymerase chain reaction for the detection of four bee viruses

J Virol Methods. 2024 Jul:328:114953. doi: 10.1016/j.jviromet.2024.114953. Epub 2024 May 15.


Viruses in the families Dicistroviridae and Iflaviridae are among the main threats to western honey bees (Apis mellifera) and native bee species. Polymerase chain reaction (PCR) is the gold standard for pathogen detection in bees. However, high throughput screening for bee virus infections in singleplex PCR reactions is cumbersome and limited by the high quantities of sample RNA required. Thus, the development of a sensitive and specific multiplex PCR detection method for screening for multiple viruses simultaneously is necessary. Here, we report the development of a one-step multiplex reverse-transcription quantitative polymerase chain reaction (RT-qPCR) assay to detect four viruses commonly encountered in pollinator species. The optimized multiplex RT-qPCR protocol described in this study allows simultaneous detection of two dicistroviruses (Israeli acute paralysis virus and Black queen cell virus) and two iflaviruses (Sacbrood virus and Deformed wing virus) with high efficiency and specificity comparable to singleplex detection assays. This assay provides a broad range of detection and quantification, and the results of virus quantification in this study are similar to those performed in other studies using singleplex detection assays. This method will be particularly useful for data generation from small-bodied insect species that yield low amounts of RNA.

Keywords: Apis mellifera; Bee virus; Honey bee; Multiplex PCR; Native pollinator.

MeSH terms

  • Animals
  • Bees / virology
  • Dicistroviridae* / genetics
  • Dicistroviridae* / isolation & purification
  • Insect Viruses / classification
  • Insect Viruses / genetics
  • Insect Viruses / isolation & purification
  • Multiplex Polymerase Chain Reaction* / methods
  • RNA Viruses* / classification
  • RNA Viruses* / genetics
  • RNA Viruses* / isolation & purification
  • RNA, Viral / genetics
  • RNA, Viral / isolation & purification
  • Real-Time Polymerase Chain Reaction / methods
  • Reverse Transcriptase Polymerase Chain Reaction / methods
  • Sensitivity and Specificity*


  • RNA, Viral

Supplementary concepts

  • Black queen cell virus
  • Deformed wing virus
  • Sacbrood virus