Potential application of CRISPR-Cas12a fluorescence assay coupled with rapid nucleic acid amplification for detection of white spot syndrome virus in shrimp

Abstract

© 2019 Elsevier B.V. White spot disease (WSD), caused by white spot syndrome virus (WSSV), is among the most severe diseases of cultivated shrimp. Here, the CRISPR-Cas12a system coupled with nucleic acid amplification was optimized for the detection of WSSV. The CRISPR-Cas12a system was used to specifically cleave the WSSV amplicons, simultaneously releasing a quenched reporter molecule resulting in fluorescence that could be detected with a simple UV transilluminator or a microplate reader. This specific cleavage accompanied by fluorescence simultaneously revealed the presence of the amplicon and confirmed its identity, preventing false positive test results from non-specific amplicons. When coupled with PCR or recombinase polymerase amplification (RPA), the Cas12a platform was capable of detecting as few as 200 copies WSSV per reaction and displayed no cross-reactivity with other shrimp DNA viruses. The method was also un-interfered by the presence of large amounts of unrelated background DNA. Moreover, the RPA-Cas12a protocol from start to finish could be performed at a constant temperature near 37 °C and required <1 h, without the need for complex equipment. Overall, our results demonstrated that the CRISPR-Cas12a method is robust, specific, confirmatory, user-friendly and potentially adaptable for in-field diagnosis of shrimp diseases.