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

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)