Wang Ziyan, Xia Boyang, Fei Tiantian, Liu Yuming, Gao Zhifeng, Shu Xiuwei, Ma Mingxiao, Fei Dongliang
Key Laboratory of Livestock Products Quality and Safety Engineering, Jinzhou Medical University, Jinzhou, 121001, China.
College of Animal Husbandry and Veterinary Medicine, Jinzhou Medical University, Jinzhou, 121001, China.
BMC Vet Res. 2025 Aug 26;21(1):524. doi: 10.1186/s12917-025-04969-5.
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.
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.
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.
作为维持农业生态系统服务和生物多样性的关键传粉者，蜜蜂为全球约35%的昆虫授粉作物介导授粉，并通过制药和食品工业中的蜂产品产生多维度的生态价值。然而，新出现的病毒病原体对蜜蜂健康构成的威胁不断升级。
为解决蜜蜂病毒性麻痹病病原体检测的技术瓶颈，本研究创新性地整合了多重逆转录聚合酶链反应（RT-PCR）扩增、侧向流动试纸条（LFD）和离心微流控芯片技术（MFCT），开发了一种能够同时鉴定四种病毒的现场四重检测平台，这四种病毒分别是慢性蜜蜂麻痹病毒（CBPV）、黑蜂王台病毒（BQCV）、残翅病毒（DWV）和以色列急性麻痹病毒（IAPV）。通过多序列比对，确定了这四种病毒的保守基因组区域，并进行系统筛选以优化引物组合，确定了引物浓度（10µM）和退火温度（55°C）等关键参数。在此基础上，通过加入化学修饰的下游引物/探针和MFCT，建立了RT-PCR-LFD-MFCT集成检测系统。实验结果表明，单病毒检测灵敏度为10²拷贝/µL，能够精确鉴定低病毒载量。该方法具有卓越的特异性，无交叉反应，临床样本验证与传统RT-qRT-PCR的一致性达到100%。
该系统具有同时多靶点检测、高特异性、处理速度快、仪器要求低、便携性和现场适用性强等特点。它为蜜蜂麻痹病的精确诊断和防控提供了有力工具，特别适用于资源有限的养蜂场和疫情爆发场景，对保障养蜂业健康具有重要的实际价值。
