PMID- 36173294 OWN - NLM STAT- MEDLINE DCOM- 20221028 LR - 20231020 IS - 2165-0497 (Electronic) IS - 2165-0497 (Linking) VI - 10 IP - 5 DP - 2022 Oct 26 TI - Differentiation of Classical Swine Fever Virus Virulent and Vaccine Strains by CRISPR/Cas13a. PG - e0089122 LID - 10.1128/spectrum.00891-22 [doi] LID - e00891-22 AB - As a notifiable terrestrial and aquatic animal disease listed by World Organisation for Animal Health (formerly the Office International des Epizooties [OIE]), classical swine fever (CSF) has caused great economic losses to the swine industry worldwide during recent decades. Differentiation of infected and vaccinated animals (DIVA) is urgent for eradication of CSF. In this study, a diagnostic platform based on CRISPR/Cas13a was established with the ability to differentiate between classical swine fever virus (CSFV) virulent and vaccine strains. In combination with reverse transcription recombinase-aided amplification (RT-RAA), the detection limit for CSFV synthetic RNA templates reached 3.0 x 10(2) copies/muL. In addition, with boiling and chemical reduction, heating unextracted diagnostic samples to obliterate nucleases (HUDSON) treatment was introduced to inactivate nucleases and release viral genome, achieving robust pretreatment of tested sample before CRISPR/Cas13a detection without the need to extract viral nucleic acids. HUDSON-RT-RAA-CRISPR/Cas13a can directly detect cell cultures of virulent Shimen strain and vaccine hog cholera lapinized virus (HCLV) strain, with the detection limit of 3.5 x 10(2) copies/muL and 1.8 x 10(2) copies/muL, respectively, which was equally sensitive to nested PCR (nPCR) and 100 times more sensitive than antigen enzyme-linked immunosorbent assay (ELISA). Meanwhile, HUDSON-RT-RAA-CRISPR/Cas13a showed no cross-reactivity with bovine viral diarrhea virus (BVDV), atypical porcine pestivirus (APPV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine epidemic diarrhea virus (PEDV), African swine fever virus (ASFV), pseudorabies virus (PRV), and porcine circovirus 2 (PCV2), exhibiting good specificity. At last, a total of 50 pig spleen samples with suspected clinical signs were also assayed with HUDSON-RT-RAA-CRISPR/Cas13a, nPCR, and antigen ELISA in parallel. HUDSON-RT-RAA-CRISPR/Cas13a showed 100.0% with nPCR and 82.0% coincident rate with antigen ELISA, respectively. IMPORTANCE Classical swine fever (CSF) is a World Organisation for Animal Health (formerly the Office International des Epizooties [OIE]) notifiable terrestrial and aquatic animal disease, causing great economic losses to the swine industry worldwide during the past decades. Due to the use of the most effective and safe attenuated live vaccine for CSF prevention, differentiation of infected and vaccinated pigs is vital work, as well as a bottleneck for eradication of CSF. Methods with the ability to precisely differentiate classical swine fever virus (CSFV) virulent strains from vaccine strain hog cholera lapinized virus (HCLV) are urgently needed. Combining the high sensitivity of isothermal recombinase-aided amplification (RAA) with the accurate molecular sensing ability of Cas13a, we presented a novel method for CSFV detection without the need to extract viral nucleic acids, which showed great advantage to traditional detection methods for precise differentiation of CSFV virulent strains and vaccine strain, providing a novel powerful tool for CSF eradication. FAU - Zhang, Yuhang AU - Zhang Y AD - International Joint Research Center of National Animal Immunology, College of Veterinary Medicine, Henan Agricultural Universitygrid.108266.b, Zhengzhou, China. FAU - Li, Qingmei AU - Li Q AD - Key Laboratory of Animal Immunology, Henan Academy of Agricultural Sciences, Zhengzhou, China. FAU - Wang, Ruining AU - Wang R AD - Henan University of Animal Husbandry and Economy, College of Veterinary Medicine, Zhengzhou, China. FAU - Wang, Li AU - Wang L AD - Key Laboratory of Animal Immunology, Henan Academy of Agricultural Sciences, Zhengzhou, China. FAU - Wang, Xun AU - Wang X AD - International Joint Research Center of National Animal Immunology, College of Veterinary Medicine, Henan Agricultural Universitygrid.108266.b, Zhengzhou, China. FAU - Luo, Jun AU - Luo J AD - Key Laboratory of Animal Immunology, Henan Academy of Agricultural Sciences, Zhengzhou, China. FAU - Xing, Guangxu AU - Xing G AD - Key Laboratory of Animal Immunology, Henan Academy of Agricultural Sciences, Zhengzhou, China. FAU - Zheng, Guanmin AU - Zheng G AD - Public Health and Preventive Medicine Teaching and Research Center, Henan University of Chinese Medicine, Zhengzhou, China. FAU - Wan, Bo AU - Wan B AD - International Joint Research Center of National Animal Immunology, College of Veterinary Medicine, Henan Agricultural Universitygrid.108266.b, Zhengzhou, China. FAU - Guo, Junqing AU - Guo J AD - Key Laboratory of Animal Immunology, Henan Academy of Agricultural Sciences, Zhengzhou, China. FAU - Zhang, Gaiping AU - Zhang G AUID- ORCID: 0000-0002-3834-9975 AD - International Joint Research Center of National Animal Immunology, College of Veterinary Medicine, Henan Agricultural Universitygrid.108266.b, Zhengzhou, China. AD - Key Laboratory of Animal Immunology, Henan Academy of Agricultural Sciences, Zhengzhou, China. AD - Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Disease and Zoonose, Yangzhou University, Yangzhou, China. LA - eng PT - Journal Article PT - Research Support, Non-U.S. Gov't DEP - 20220929 PL - United States TA - Microbiol Spectr JT - Microbiology spectrum JID - 101634614 RN - 0 (Viral Vaccines) RN - 0 (Recombinases) RN - 0 (Nucleic Acids) RN - 63231-63-0 (RNA) SB - IM MH - Swine MH - Animals MH - *Classical Swine Fever Virus/genetics MH - *Classical Swine Fever/diagnosis/prevention & control/genetics MH - Clustered Regularly Interspaced Short Palindromic Repeats MH - *African Swine Fever Virus/genetics MH - *Viral Vaccines MH - Sensitivity and Specificity MH - Recombinases/genetics MH - *Nucleic Acids MH - RNA PMC - PMC9603908 OTO - NOTNLM OT - CRISPR/Cas13a OT - classical swine fever virus OT - differentiation of infected and vaccinated animals OT - recombinase-aided amplification COIS- The authors declare no conflict of interest. EDAT- 2022/09/30 06:00 MHDA- 2022/10/29 06:00 PMCR- 2022/09/29 CRDT- 2022/09/29 10:05 PHST- 2022/09/30 06:00 [pubmed] PHST- 2022/10/29 06:00 [medline] PHST- 2022/09/29 10:05 [entrez] PHST- 2022/09/29 00:00 [pmc-release] AID - 00891-22 [pii] AID - spectrum.00891-22 [pii] AID - 10.1128/spectrum.00891-22 [doi] PST - ppublish SO - Microbiol Spectr. 2022 Oct 26;10(5):e0089122. doi: 10.1128/spectrum.00891-22. Epub 2022 Sep 29.