Construction of a portable multiplex detection system for four bee viral paralysis diseases based on RT-PCR-microfluidic chip integrated technology

Abstract Background As crucial pollinators sustaining agricultural ecosystem services and biodiversity, bees mediate pollination for approximately 35% of global insect-pollinated crops and generate multidimensional ecological value through apicultural products in the pharmaceutical and food industries. However, emerging viral pathogens pose escalating threats to bee health. Results To address the technical bottlenecks in pathogen detection for viral paralysis disease in bees, this study innovatively integrated multiplex RT-PCR amplification, lateral flow dipstick (LFD), and centrifugal microfluidic chip technology (MFCT) to develop an on-site quadruple detection platform capable of simultaneously identifying four viruses: Chronic Bee Paralysis Virus (CBPV), Black Queen Cell Virus (BQCV), Deformed Wing Virus (DWV), and Israeli Acute Paralysis Virus (IAPV). Through multiple sequence alignment, conserved genomic regions of the four viruses were identified, and systematic screening was performed to optimize primer combinations, with critical parameters such as primer concentration (10 µM) and annealing temperature (55 °C) determined. Building on this, a RT-PCR-LFD-MFCT integrated detection system was established by incorporating chemically modified downstream primers/probes and MFCT. Experimental results demonstrated a sensitivity of 10² copies/µL for single-virus detection, enabling precise identification of low viral loads. The method exhibited exceptional specificity with no cross-reactivity, and clinical sample validation achieved 100% concordance with conventional RT-qRT-PCR. Conclusions This system features simultaneous multi-target detection, high specificity, rapid processing, minimal instrumentation requirements, portability, and field applicability. It provides a robust tool for precise diagnosis and control of bee paralysis diseases, particularly suitable for resource-limited apiaries and outbreak scenarios, demonstrating significant practical value for safeguarding apicultural health.