Abstract:Spring viremia of carp virus (SVCV) is highly pathogenic in cyprinid fishes, and spring viremia of carp (SVC) has been listed as a notifiable disease by the World Organization for Animal Health. Spring viremia of common carp (Cyprinus carpio) is a viral infectious disease prevalent in Europe, Asia, and North America. In 2002, SVC was detected for the first time in China and spread rapidly throughout the country, posing a huge threat to the carp farming industry in China. It was listed as a second-class animal disease. So far, early, rapid, and accurate diagnosis remains an important means to control its spread and prevalence. At present, commonly used methods to detect SVCV require specific amplification equipment and temperature cycles and are prone to false negatives. Moreover, they have high equipment and personnel requirements and a long detection cycle. Fast and effective daily testing is also difficult to complete. Recombinase-aided amplification (RAA) is a rapid amplification technique of nucleic acid at constant temperature, which can achieve nucleic acid amplification at constant temperature and is easy to operate. Regular clustered interspaced short palindromic repeats (CRISPR) and their associated proteins (Cas) systems, such as CRISPR-Cas12 and CRISPR-Cas13, are combined with isothermal amplification to improve the specificity and sensitivity of RNA virus detection. This technology is characterized by constant temperature, fast reaction speed, and miniaturization, and it is suitable for rapid diagnosis and regular monitoring in the field. As an SVC international reference laboratory recognized by WOAH, this research team has been cooperating with the SVC reference laboratory in the UK to carry out the screening, comparison, and optimization of nucleic acid rapid detection. Based on the CRISPR/Cas13a system and recombinase-mediated isothermal nucleic acid amplification technology, RAA amplification primers and corresponding crRNA primers were designed for the highly conserved region of SVCV polymerase L gene by aligning the whole gene sequence of SVCV registered on GenBank, and a preliminary RAA-CRISPR/Cas13a detection method was established for the rapid detection of SVCV in the field. It can cover four genotypes of SVCV (Ia, Ib, Ic, Id). In accordance with the detection and verification procedures recommended by the World Organization for Animal Health, results confirmed that some of the detection results were reproducible, the minimum detection concentration was 115 copies/μL, and it did not cross-react with other pathogens. The detection of 60 samples isolated and stored in the laboratory for the monitoring of inbound and outbound aquatic animal diseases, the monitoring of major aquatic animal diseases in China, and the challenge experiment indicated that the results of RAA-CRISPR/Cas13a detection are consistent with those of nested fluorescent RT-PCR. The results of RT-PCR and virus isolation and culture were also consistent, and the diagnostic sensitivity and specificity were both 100%. Under the same conditions, 20 samples were tested in four laboratories, and the results of the four laboratories were consistent. The results proved the good reproducibility of the study. This study is the first to use the CRISPR-Cas13a system for SVCV detection. The developed method could be applied in the rapid diagnosis and prevention of SVCV.
