Abstract: To establish a highly sensitive detection method for sacbrood virus（SBV） in bees，the predominant circulating strain（GenBank accession no. MZ821922. 1）of SBV in Apis cerana cerana and Apis mellifera ligustica from Mengzi City，Yunnan Province，was identified by RT⁃PCR sequencing，and specific primers were designed based on the conserved regions of this strain. The total RNA of the samples was extracted and the cDNA was obtained by reverse transcription.The RT⁃qPCR detection method was established by SYBR Green Ⅰ dye method，the specificity，sensitivity，repeatability and practicality were verified.The results demonstrated the high quality of the designed primers.The concentration of SBV positive plasmid standard was 2.62×10^¹⁰ copies/μL，and the linear range was 2.62×10^³—2.62×1010 copies/μL（R^²=0.999 1），and the amplification efficiency was 102.6%.Furthermore，the assay was proved to be highly sensitive（detection limit：2.62×10^⁰ copies/μL），specific（no cross⁃reactivity with other common honeybee viruses），and reproducible（intra⁃and inter⁃assay CV<1.19%）.Two⁃step RT⁃qPCR and conventional RT⁃PCR were performed on bee samples from Mengzi City and Gongshan County，Yunnan Province. It was found that the detection sensitivity of two⁃step RT⁃PCR was significantly higher than that of RT⁃PCR：the positive detection rate of total samples（56.1% vs 11.5%），Chinese bee in Gongshan County（46.2% vs 5.4%），Chinese bee in Mengzi City（80. 0% vs 250%）and Italian bee in Mengzi City（53.3% vs 13.3%）.These findings further demonstrated the clinical applicability of the established method. In conclusion，the developed two⁃step SYBR Green Ⅰ⁃based RT⁃qPCR assay for SBV is sensitive and reliable，and can provide effective technical support for the rapid detection，early diagnosis，and epidemiological surveillance of SBV in China.

Key words: Apis cerena cerena, Apis mellifera ligustica, Sacbrood virus, Two?step fluorescence quantitative PCR, SYBR dye method

摘要： 为建立高灵敏度的蜜蜂囊状幼虫病毒（SBV）检测方法，通过反转录聚合酶链式反应（RT-PCR）测序确定了云南省蒙自市的中华蜜蜂与意大利蜜蜂蜂群中SBV 的优势流行株（GenBank 登录号：MZ821922.1），针对其保守序列设计引物。提取样品总RNA并经反转录获得cDNA后，采用SYBR GreenⅠ染料法建立反转录实时荧光定量聚合酶链式反应（RT-qPCR）检测方法，并对该方法的特异性、灵敏度、重复性及实用性进行验证。结果表明，所设计引物特异性良好；构建的SBV阳性质粒标准品浓度为2.62×10^¹⁰ copies/μL，在2.62×10^³—2.62×10^¹⁰ copies/μL浓度范围内线性关系良好（R^²=0.999 1），扩增效率为102.6%。该方法灵敏度高（检出限为2.62×100 copies/μL）、特异性强（与蜜蜂其他3种常见病毒无交叉反应），且重复性好（组内、组间变异系数均低于1.19%）。对云南省蒙自市与贡山县蜂样进行两步法RT-qPCR和RT-PCR检测，发现两步法RT-qPCR的检测灵敏度明显高于RT-PCR：总样本阳性检出率（56.1% vs 11.5%）、贡山县中华蜜蜂（46.2% vs 5.4%）、蒙自市中华蜜蜂（80.0% vs 25.0%）及蒙自市意大利蜜蜂（53.3% vs 13.3%）。综上，所建立的SBV两步法荧光定量PCR（SYBR Green Ⅰ）检测方法灵敏、可靠，可为我国SBV的快速检测、早期诊断与流行病学监测提供有效技术支持。

关键词: 中华蜜蜂, 意大利蜜蜂, 囊状幼虫病毒, 两步法荧光定量PCR, SYBR染料法