Establishment of Fluorescence Quantitative PCR Method for Detection of Sacbrood Virus in Honeybee

WANG Hongkun; ZHANG Xuan; ZONG Deqin; LI Yushi; LÜ Mingkui; ZHAO Hongmu

doi:10.15933/j.cnki.1004-3268.2025.12.014

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 2.62×1010 copies/µL,and the linear range was 2.62×103-2.62×1010 copies/µL(R2=0.999 1),and the amplification efficiency was 102.6%.Furthermore,the assay was proved to be highly sensitive(detection limit:2.62×10⁰ 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 25.0%)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.