Rapid detection of Chinese sacbrood virus via CRISPR-Cas13a-based lateral flow strips

Abstract

Sacbrood virus (SBV) is one of the most pathogenic honeybee viruses with host specificity and regional variation. The SBV strain infecting the Chinese honeybee (Apis cerana) is known as Chinese sacbrood virus (CSBV). The extensively used CSBV detection methods require professionals and expensive equipment; thus, they are unsuitable for rapid onsite CSBV detection. To achieve early and rapid detection of CSBV, we developed a lateral flow detection (LFD) strip method for CSBV detection via clustered regularly interspaced short palindromic repeats (CRISPR) and the Cas13a technique. On the basis of the conserved CSBV VP2 gene nucleotide region, we designed 3 recombinant enzyme-assisted amplification (RAA) primer pairs and prepared 3 corresponding crRNAs. We investigated key performance metrics, including the sensitivity, specificity, and accuracy of LFD strips. The results demonstrated that the LFD strip based on the optimal combination (primer 2+crRNA 2) presented the lowest detection limit (2.80×10¹ copies/μL), and this strip could complete CSBV detection within 1 h. Furthermore, this strip exhibited excellent detection specificity, with no cross-reactivity with four other honeybee viruses. A test of 100 clinical samples indicated the feasibility of the LFD method for CSBV detection. A comparison of various CSBV detection methods revealed that the CRISPR-Cas13a-based LFD method was more accurate, efficient, and sensitive than the other methods were, indicating great application prospects in onsite CSBV detection. Our developed method is highly important for preventing and controlling CSBV infection as well as maintaining honeybee health.

Keywords

Chinese sacbrood virus / CRISPR-Cas13a / RAA / Lateral flow strip (LFS) / Rapid detection

References

[1] AbudayyehOO, GootenbergJS, EssletzbichlerP, HanS, JoungJ, BelantoJJ, VerdineV, CoxDBT, KellnerMJ, RegevA, LanderES, VoytasDF, TingAY, ZhangF. RNA targeting with CRISPR–Cas13. Nature, 2017, 550(7675): 280-284.

[2] Agarwal N., Gupta R. 2021. Chapter Two - History, evolution and classification of CRISPR‒Cas associated systems, p 11–76. In Singh V (ed), Progress in Molecular Biology and Translational Science, vol 179. Academic Press.

[3] ChenC, RidzonDA, BroomerAJ, ZhouZ, LeeDH, NguyenJT, BarbisinM, XuNL, MahuvakarVR, AndersenMR, et al.. Real-time quantification of microRNAs by stem–loop RT–PCR. Nucleic Acids Research, 2005, 33(20): e179-e179.

[4] FreibergM, JongDD, MessageD, Cox-FosterD. First report of sacbrood virus in honey bee (Apis mellifera) colonies in Brazil. Genetics and Molecular Research, 2012, 11(3): 3310-3314.

[5] GootenbergJS, AbudayyehOO, LeeJW, EssletzbichlerP, DyAJ, JoungJ, VerdineV, DonghiaN, DaringerNM, FreijeCA, et al.. Nucleic acid detection with CRISPR-Cas13a/C2c2. Science, 2017, 356(6336): 438-442.

[6] Kukielka D., Perez A.M., Higes M., Bulboa M.dC., Sánchez-Vizcaíno J.M. 2008. Analytical sensitivity and specificity of a RT‒PCR for the diagnosis and characterization of the spatial distribution of three Apis mellifera viral diseases in Spain. Apidologie, 39 (6): 607–617. https://doi.org/10.1051/apido:2008040.

[7] LiY, LiS, WangJ, LiuG. CRISPR/Cas systems toward next-generation biosensing. Trends in Biotechnology, 2019, 37(7): 730-743.

[8] LiM, SunL, MaY, FeiD, MaM. Development of a sandwich ELISA for the detection of Chinese sacbrood virus infection. Archives of Virology, 2020, 165(7): 1551-1556.

[9] LiljasL, TateJ, LinT, ChristianP, JohnsonJE. Evolutionary and taxonomic implications of conserved structural motifs between picornaviruses and insect picorna-like viruses. Archives of Virology, 2002, 147(1): 59-84.

[10] LiuY, LiuH, YuG, SunW, AizazM, YangG, ChenL. One-tube RPA-CRISPR Cas12a/Cas13a rapid detection of methicillin-resistant Staphylococcus aureus. Analytica Chimica Acta, 2023, 1278. 341757

[11] MaM, MaC, LiM, WangS, YangS, WangS. Loop-mediated isothermal amplification for rapid detection of Chinese sacbrood virus. Journal of Virological Methods, 2011, 176(1): 115-119.

[12] MingxiaoM, JinhuaL, YingjinS, LiL, YongfeiL. TaqMan MGB probe fluorescence real-time quantitative PCR for rapid detection of Chinese sacbrood virus. PLoS ONE, 2013, 82. e52670

[13] Mingxiao M., Ming L., Jian C., Song Y., Shude W., Pengfei L. 2011. Molecular and biological characterization of Chinese sacbrood virus LN isolate. International Journal of bGenomics, 2011 (1): 409386. https://doi.org/10.1155/2011/409386.

[14] PrakashS, JainA, JainB. Development of novel triplex single-step real-time PCR assay for detection of Hepatitis Virus B and C simultaneously. Virology, 2016, 492: 101-107.

[15] Procházková M., Füzik T., Škubník K., Moravcová J., Ubiparip Z., Přidal A., Plevka P. 2018. Virion structure and genome delivery mechanism of sacbrood honeybee virus. Proceedings of the National Academy of Sciences, 115 (30): 7759–7764. https://doi.org/10.1073/pnas.1722018115.

[16] Ren W., Zhou Y., Li H., Shang Y., Zhang X., Yuan J., Li S., Li C., Pang Y. 2023. Development and clinical evaluation of a CRISPR/Cas13a-based diagnostic test to detect Mycobacterium tuberculosis in clinical specimens. Frontiers in Microbiology, 14: https://doi.org/10.3389/fmicb.2023.1117085.

[17] ShanL, LiuhaoW, JunG, YujieT, YanpingC, JieW, JilianL. Chinese sacbrood virus infection in Asian honey bees (Apis cerana cerana) and host immune responses to the virus infection. Journal of Invertebrate Pathology, 2017, 150: 63-69.

[18] WangL, ZhouJ, WangQ, WangY, KangC. Rapid design and development of CRISPR-Cas13a targeting SARS-CoV-2 spike protein. Theranostics, 2021, 11(2): 649-664.

[19] Wang J., Zhu X., Yin D., Cai C., Liu H., Yang Y., Guo Z., Yin L., Shen X., Dai Y., Pan X.. 2023. Rapid and easy-read porcine circovirus type 4 Ddetection with CRISPR–Cas13a-based lateral flow strip. Microorganisms. 11 (2): https://doi.org/10.3390/microorganisms11020354.

[20] WeiN, ZhengB, NiuJ, ChenT, YeJ, SiY, CaoS. Rapid detection of genotype II African swine fever virus using CRISPR Cas13a-based lteral flow strip. Viruses, 2022, 142179.

[21] ZhangQ, YangY, LiangY, LuX, ZhangJ. Study on structure of the Chinese sacbrood virus' nucleic acid. Journal of Chinese Electronic Microscopy Society, 2002, 21(3): 331-334

[22] ZhangL, YangL, LiH, YuY, ZhaiJ. Rapid detection of insect pest with CRISPR-Cas13a-Based Lateral Flow Strip: Using Locusta migratoria manilensis. Journal of Applied Entomology, 2023, 147(6): 416-422.

Funding

the National Key Research and Development Program of China(2022YFD1600202)

the Lush Mountain Public Welfare Protection Action of the China Environmental Protection Foundation(CEPFQS202169-14)

RIGHTS & PERMISSIONS

The Author(s)