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

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