The infection of Bombyx mori nucleopolyhedrovirus (BmNPV) is one of the main causes of economic losses in sericulture. Thus, it is essential to establish rapid and effective method for BmNPV detection. In the present study, we have developed a recombinase-aided amplification (RAA) to amplify the BmNPV genomic DNA at 37 °C within 30 min, and achieved a rapid detection method by coupling with a lateral flow dipstick (LFD). The RAA-LFD method had a satisfactory detection limit of 6 copies/μL of recombinant plasmid pMD19-T-IE1, and BmNPV infection of silkworm can be detected 12 h post-infection. This method was highly specific for BmNPV, and without cross-reactivity to other silkworm pathogens. In contrast to conventional polymerase chain reaction (PCR), the RAA-LFD assay showed higher sensitivity, cost-saving, and especially is apt to on-site detection of BmNPV infection in the sericulture production.

Institute of Sericulture Chinese Academy of Agricultural Sciences Zhenjiang China

School of Biotechnology Jiangsu University of Science and Technology Zhenjiang China

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Bao Y, Tang X, Lv Z, Wang X, Tian C, Xu Y, Zhang C (2009) Gene expression profiling of resistant and susceptible Bombyx mori strains reveals nucleopolyhedrovirus-associated variations in host gene transcript levels. Genomics 94:138–145. https://doi.org/10.1016/j.ygeno.2009.04.003 PubMed DOI

Borchers A, Pieler T (2023) Programming pluripotent precursor cells derived from Xenopus embryos to generate specific tissues and organs. Genes (basel) 1:413–426. https://doi.org/10.3390/genes1030413 DOI

Chen C, Li XN, Li GX, Zhao L, Duan SX, Yan TF, Feng ZS, Ma XJ (2018) Use of a rapid reverse-transcription recombinase aided amplification assay for respiratory syncytial virus detection. Diagn Microbiol Infect Dis 90:90–95. https://doi.org/10.1016/j.diagmicrobio.2017.10.005 PubMed DOI

Chen W, Fan J, Li Z, Zhang Y, Qin Y, Wu K, Li X, Li Y, Fan S, Zhao M (2021) Development of recombinase aided amplification combined with disposable nucleic acid test strip for rapid detection of Porcine circovirus type 2. Front Vet Sci 8:676294. https://doi.org/10.3389/fvets.2021.676294 PubMed DOI PMC

Chen Y, Zong N, Ye F, Mei Y, Qu J, Jiang X (2023) Dual-CRISPR/Cas12a-assisted RT-RAA for ultrasensitive SARS-CoV-2 detection on automated centrifugal microfluidics. Anal Chem 94:9603–9609. https://doi.org/10.1021/acs.analchem.2c00638 DOI

Fan X, Li L, Zhao Y, Liu Y, Liu C, Wang Q, Dong Y, Wang S, Chi T, Song F, Sun C, Wang Y, Ha D, Zhao Y, Bao J, Wu X, Wang Z (2020) Clinical validation of two recombinase-based isothermal amplification assays (RPA/RAA) for the rapid detection of African swine fever virus. Front Microbiol 11:1696. https://doi.org/10.3389/fmicb.2020.01696 PubMed DOI PMC

Fu Z, He X, Cai S, Liu H, He X, Li M, Lu X (2016) Quantitative PCR for detection of Nosema bombycis in single silkworm eggs and newly hatched larvae. J Microbiol Methods 120:72–78. https://doi.org/10.1016/j.mimet.2015.12.003 PubMed DOI

Gill P, Ghaemi A (2008) Nucleic acid isothermal amplification technologies: a review. Nucleosides Nucleotides Nucleic Acids 27:224–243. https://doi.org/10.1080/15257770701845204 PubMed DOI

Glais L, Jacquot E (2015) Detection and characterization of viral species/subspecies using isothermal recombinase polymerase amplification (RPA) assays. Methods Mol Biol 1302:207–225. https://doi.org/10.1007/978-1-4939-2620-6_16 PubMed DOI

Gomi S, Majima K, Maeda S (1999) Sequence analysis of the genome of Bombyx mori nucleopolyhedrovirus. J Gen Virol 80:1323–1337. https://doi.org/10.1099/0022-1317-80-5-1323 PubMed DOI

Gui L, Zhao Y, Xu D, Li X, Luo J, Zhou W, Li M (2022) Quick detection of Carassius auratus herpesvirus (CaHV) by recombinase-aid amplification lateral flow dipstick (RAA-LFD) method. Front Cell Infect Microbiol 12:981911. https://doi.org/10.3389/fcimb.2022.981911 PubMed DOI PMC

Guo Z, Xing G, Li P, Jin Q, Lu Q, Zhang G (2023) Development and application of a recombinase-aided amplification and lateral flow assay for rapid detection of pseudorabies virus from clinical crude samples. Int J Biol Macromol 1:646–652. https://doi.org/10.1016/j.ijbiomac.2022.10.153 DOI

He Z, Ni Q, Song Y, Wang R, Tang Y, Wu Y, Liu L, Bao J, Chen J, Long M, Wei J, Li C, Li T, Zhou Z, Pan G (2019) Development of a nucleic acid lateral flow strip for rapid, visual detection of Nosema bombycis in silkworm eggs. J Invertebr Pathol 164:59–65. https://doi.org/10.1016/j.jip.2019.04.004 PubMed DOI

Huang W, Wei H, Wang C, Wang J, Chen L, Chen W, Liu Y, Zheng Y, Lin M (2023) Establishment and preliminary evaluation of a fluorescent recombinase-aided amplification/CRISPR-Cas12a system for rapid detection of Plasmodium falciparum. Zhongguo Xue Xi Chong Bing Fang Zhi Za Zhi 35:38–43. https://doi.org/10.16250/j.32.1374.2022240

Jiang L, Xia Q (2014) The progress and future of enhancing antiviral capacity by transgenic technology in the silkworm Bombyx mori. Insect Biochem Mol Biol 48:1–7. https://doi.org/10.1016/j.ibmb.2014.02.003 PubMed DOI

Kang W, Suzuki M, Zemskov E, Okano K, MaedA S (1999) Characterization of baculovirus repeated open reading frames (bro) in Bombyx mori nucleopolyhedrovirus. J Virol 73:10339–10345. https://doi.org/10.1128/JVI.73.12.10339-10345.1999 PubMed DOI PMC

Kiatpathomchai W, Jaroenram W, Arunrut N, Jitrapakdee S, Flegel TW (2008) Shrimp Taura syndrome virus detection by reverse transcription loop-mediated isothermal amplification combined with a lateral flow dipstick. J Virol Methods 153:214–217. https://doi.org/10.1016/j.jviromet.2008.06.025 PubMed DOI

Kursa O, Wozniakowski G, Tomczyk G, Sawicka A, Minta Z (2015) Rapid detection of Mycoplasma synoviae by loop-mediated isothermal amplification. Arch Microbiol 197:319–325. https://doi.org/10.1007/s00203-014-1063-2 PubMed DOI

Piepenburg O, Williams CH, Stemple DL, Armes NA (2006) DNA detection using recombination proteins. PLoS Biol 4:1115–1121. https://doi.org/10.1371/journal.pbio.0040204 DOI

Rahman MM, Gopinathan KP (2004) Systemic and in vitro infection process of Bombyx mori nucleopolyhedrovirus. Virus Res 101:109–118. https://doi.org/10.1016/j.virusres.2003.12.027 PubMed DOI

Ravikumar G, Raje Urs S, Vijaya Prakash NB, Rao CG, Vardhana KV (2011) Development of a multiplex polymerase chain reaction for the simultaneous detection of microsporidians, nucleopolyhedrovirus, and densovirus infecting silkworms. J Invertebr Pathol 107:193–197. https://doi.org/10.1016/j.jip.2011.04.009 PubMed DOI

Shen XX, Qiu FZ, Shen LP, Yan TF, Zhao MC, Qi JJ, Chen C, Zhao L, Wang L, Feng ZS, Ma XJ (2019) A rapid and sensitive recombinase aided amplification assay to detect hepatitis B virus without DNA extraction. BMC Infect Dis 19:229. https://doi.org/10.1186/s12879-019-3814-9 PubMed DOI PMC

Singpanomchai N, Akeda Y, Tomono K, Tamaru A, Santanirand P, Ratthawongjirakul P (2019) Naked eye detection of the Mycobacterium tuberculosis complex by recombinase polymerase amplification-SYBR green I assays. J Clin Lab Anal 33:e22655. https://doi.org/10.1002/jcla.22655 PubMed DOI

Wang F, Liang Q, Lv R, Ahmad S, Bano M, Weng G, Wen R (2023) Optimization and validation of reverse transcription recombinase-aided amplification (RT-RAA) for Sorghum mosaic virus detection in sugarcane. Pathogens 12. https://doi.org/10.3390/pathogens12081055

Wang W, Wang C, Bai Y, Zhang P, Yao S, Liu J, Zhang T (2020a) Establishment of reverse transcription recombinase-aided amplification-lateral-flow dipstick and real-time fluorescence-based reverse transcription recombinase-aided amplification methods for detection of the Newcastle disease virus in chickens. Poult Sci 99:3393–3401. https://doi.org/10.1016/j.psj.2020.03.018 PubMed DOI PMC

Wang W, Wang C, Zhang P, Yao S, Liu J, Zhai X, Zhang T (2020b) Reverse transcription recombinase-aided amplification assay combined with a lateral flow dipstick for detection of avian infectious bronchitis virus. Poult Sci 99:89–94. https://doi.org/10.3382/ps/pez559 PubMed DOI

Wang XY, Shao ZM, Zhang YJ, Vu TT, Wu YC, Xu JP, Deng MJ (2019) A PubMed DOI

Wu Q, Xu X, Chen Q, Zuo K, Zhou Y, Zhang Z, Kan Y, Yao L, Ji J, Bi Y, Xie Q (2019) Rapid and visible detection of Mycoplasma synoviae using a novel polymerase spiral reaction assay. Poult Sci 98:5355–5360. https://doi.org/10.3382/ps/pez356 PubMed DOI

Xiong Y, Luo Y, Li H, Wu W, Ruan X, Mu X (2020) Rapid visual detection of dengue virus by combining reverse transcription recombinase-aided amplification with lateral-flow dipstick assay. Int J Infect Dis 95:406–412. https://doi.org/10.1016/j.ijid.2020.03.075 PubMed DOI

Yan W, Shen Z, Tang X, Xu L, Li Q, Yue Y, Xiao S, Fu X (2014) Detection of Nosema bombycis by FTA cards and loop-mediated isothermal amplification (LAMP). Curr Microbiol 69:532–540. https://doi.org/10.1007/s00284-014-0619-3 PubMed DOI

Zhang P, Wang J, Lu Y, Hu Y, Xue R, Cao G, Gong C (2014) Resistance of transgenic silkworm to BmNPV could be improved by silencing ie-1 and lef-1 genes. Gene Ther 21:81–88. https://doi.org/10.1038/gt.2013.60 PubMed DOI

Zhang Y, Zhu F, Shao Y, Su Z, Ni J, Bai X (2021) Genomic sequence analysis of Bombyx mori nucleopolyhedrovirus isolated from Yunnan sericulture region, China. Indian J Microbiol 61:383–390. https://doi.org/10.1007/s12088-021-00947-1 PubMed DOI PMC

Zhou Y, Wu J, Lin F, Chen N, Yuan S, Ding L, Gao L, Hang B (2015) Rapid detection of Bombyx mori nucleopolyhedrovirus (BmNPV) by loop-mediated isothermal amplification assay combined with a lateral flow dipstick method. Mol Cell Probes 29:389–395. https://doi.org/10.1016/j.mcp.2015.09.003 PubMed DOI