African swine fever (ASF) is an infectious disease caused by African swine fever virus (ASFV) with clinical symptoms of high fever, hemorrhages and high mortality rate, posing a threat to the global swine industry and food security. Quarantine and control of ASFV is crucial for preventing swine industry from ASFV infection. In this study, a recombinase polymerase amplification (RPA)-CRISPR-based nucleic acid detection method was developed for diagnosing ASF. As a highly sensitive method, RPA-CRISPR can detect even a single copy of ASFV plasmid and genomic DNA by determining fluorescence signal induced by collateral cleavage of CRISPR-lwCas13a (previously known as C2c2) through quantitative real-time PCR (qPCR) and has the same or even higher sensitivity than the traditional qPCR method. A lateral flow strip was developed and used in combination with RPA-CRISPR for ASFV detection with the same level of sensitivity of TaqMan qPCR. Likewise, RPA-CRISPR is capable of distinguishing ASFV genomic DNA from viral DNA/RNA of other porcine viruses without any cross-reactivity. This diagnostic method is also available for diagnosing ASFV clinical DNA samples with coincidence rate of 100% for both ASFV positive and negative samples. RPA-CRISPR has great potential for clinical quarantine of ASFV in swine industry and food security.
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This research was supported by the National Natural Science Foundation of China (31522057 and 31872451 to LZ; 31720103917 and 31872452 to ZF).
Conflicts of interest
The authors declare that they have no conflict of interest.
Animal and Human Rights Statement
This study was performed in accordance with recommendations in the care and use of laboratory animals of the Ministry of Science and Technology of China and was approved by the Scientific Ethics Committee of Huazhong Agricultural University (permit number HZAUSW-2019–007 approved on May 16, 2019).
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Ren, M., Mei, H., Zhou, M. et al. Development of A Super-Sensitive Diagnostic Method for African Swine Fever Using CRISPR Techniques. Virol. Sin. 36, 220–230 (2021). https://doi.org/10.1007/s12250-020-00323-1
- African swine fever (ASFV)
- Recombinase polymerase amplification (RPA)
- Sensitive diagnosis