Differentiation of Classical Swine Fever Virus Virulent and Vaccine Strains by CRISPR/Cas13a. 2022

Yuhang Zhang, and Qingmei Li, and Ruining Wang, and Li Wang, and Xun Wang, and Jun Luo, and Guangxu Xing, and Guanmin Zheng, and Bo Wan, and Junqing Guo, and Gaiping Zhang
International Joint Research Center of National Animal Immunology, College of Veterinary Medicine, Henan Agricultural Universitygrid.108266.b, Zhengzhou, China.

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 × 102 copies/μL. 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 × 102 copies/μL and 1.8 × 102 copies/μL, 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.

UI MeSH Term Description Entries
D009696 Nucleic Acids High molecular weight polymers containing a mixture of purine and pyrimidine nucleotides chained together by ribose or deoxyribose linkages. Nucleic Acid,Acid, Nucleic,Acids, Nucleic
D006691 Classical Swine Fever An acute, highly contagious disease affecting swine of all ages and caused by the CLASSICAL SWINE FEVER VIRUS. It has a sudden onset with high morbidity and mortality. Hog Cholera,Swine Fever,Swine Fever, Classical,Cholera, Hog
D006692 Classical Swine Fever Virus A species of the PESTIVIRUS genus causing exceedingly contagious and fatal hemorrhagic disease of swine. Hog Cholera Virus,Swine Fever Virus,Swine Fever Virus, Classical,Cholera Virus, Hog
D000358 African Swine Fever Virus The lone species of the genus Asfivirus. It infects domestic and wild pigs, warthogs, and bushpigs. Disease is endemic in domestic swine in many African countries and Sardinia. Soft ticks of the genus Ornithodoros are also infected and act as vectors. Wart-Hog Disease Virus,Virus, Wart-Hog Disease,Wart Hog Disease Virus
D000818 Animals Unicellular or multicellular, heterotrophic organisms, that have sensation and the power of voluntary movement. Under the older five kingdom paradigm, Animalia was one of the kingdoms. Under the modern three domain model, Animalia represents one of the many groups in the domain EUKARYOTA. Animal,Metazoa,Animalia
D012313 RNA A polynucleotide consisting essentially of chains with a repeating backbone of phosphate and ribose units to which nitrogenous bases are attached. RNA is unique among biological macromolecules in that it can encode genetic information, serve as an abundant structural component of cells, and also possesses catalytic activity. (Rieger et al., Glossary of Genetics: Classical and Molecular, 5th ed) RNA, Non-Polyadenylated,Ribonucleic Acid,Gene Products, RNA,Non-Polyadenylated RNA,Acid, Ribonucleic,Non Polyadenylated RNA,RNA Gene Products,RNA, Non Polyadenylated
D012680 Sensitivity and Specificity Binary classification measures to assess test results. Sensitivity or recall rate is the proportion of true positives. Specificity is the probability of correctly determining the absence of a condition. (From Last, Dictionary of Epidemiology, 2d ed) Specificity,Sensitivity,Specificity and Sensitivity
D013552 Swine Any of various animals that constitute the family Suidae and comprise stout-bodied, short-legged omnivorous mammals with thick skin, usually covered with coarse bristles, a rather long mobile snout, and small tail. Included are the genera Babyrousa, Phacochoerus (wart hogs), and Sus, the latter containing the domestic pig (see SUS SCROFA). Phacochoerus,Pigs,Suidae,Warthogs,Wart Hogs,Hog, Wart,Hogs, Wart,Wart Hog
D014765 Viral Vaccines Suspensions of attenuated or killed viruses administered for the prevention or treatment of infectious viral disease. Viral Vaccine,Vaccine, Viral,Vaccines, Viral
D045522 Recombinases A broad category of enzymes that are involved in the process of GENETIC RECOMBINATION. Recombinase,Resolvase,Resolvases

Related Publications

Yuhang Zhang, and Qingmei Li, and Ruining Wang, and Li Wang, and Xun Wang, and Jun Luo, and Guangxu Xing, and Guanmin Zheng, and Bo Wan, and Junqing Guo, and Gaiping Zhang
June 2000, Veterinary microbiology,
Yuhang Zhang, and Qingmei Li, and Ruining Wang, and Li Wang, and Xun Wang, and Jun Luo, and Guangxu Xing, and Guanmin Zheng, and Bo Wan, and Junqing Guo, and Gaiping Zhang
March 2011, Virology journal,
Yuhang Zhang, and Qingmei Li, and Ruining Wang, and Li Wang, and Xun Wang, and Jun Luo, and Guangxu Xing, and Guanmin Zheng, and Bo Wan, and Junqing Guo, and Gaiping Zhang
April 2024, iScience,
Yuhang Zhang, and Qingmei Li, and Ruining Wang, and Li Wang, and Xun Wang, and Jun Luo, and Guangxu Xing, and Guanmin Zheng, and Bo Wan, and Junqing Guo, and Gaiping Zhang
July 2018, Vaccine,
Yuhang Zhang, and Qingmei Li, and Ruining Wang, and Li Wang, and Xun Wang, and Jun Luo, and Guangxu Xing, and Guanmin Zheng, and Bo Wan, and Junqing Guo, and Gaiping Zhang
January 1998, Virus genes,
Yuhang Zhang, and Qingmei Li, and Ruining Wang, and Li Wang, and Xun Wang, and Jun Luo, and Guangxu Xing, and Guanmin Zheng, and Bo Wan, and Junqing Guo, and Gaiping Zhang
November 1995, Veterinary microbiology,
Yuhang Zhang, and Qingmei Li, and Ruining Wang, and Li Wang, and Xun Wang, and Jun Luo, and Guangxu Xing, and Guanmin Zheng, and Bo Wan, and Junqing Guo, and Gaiping Zhang
January 2017, Frontiers in microbiology,
Yuhang Zhang, and Qingmei Li, and Ruining Wang, and Li Wang, and Xun Wang, and Jun Luo, and Guangxu Xing, and Guanmin Zheng, and Bo Wan, and Junqing Guo, and Gaiping Zhang
December 2003, Virus research,
Yuhang Zhang, and Qingmei Li, and Ruining Wang, and Li Wang, and Xun Wang, and Jun Luo, and Guangxu Xing, and Guanmin Zheng, and Bo Wan, and Junqing Guo, and Gaiping Zhang
July 2008, Journal of veterinary diagnostic investigation : official publication of the American Association of Veterinary Laboratory Diagnosticians, Inc,
Yuhang Zhang, and Qingmei Li, and Ruining Wang, and Li Wang, and Xun Wang, and Jun Luo, and Guangxu Xing, and Guanmin Zheng, and Bo Wan, and Junqing Guo, and Gaiping Zhang
January 2022, Frontiers in cellular and infection microbiology,
Copied contents to your clipboard!