|WANG, DEGUO - Xuchang University|
|YU, JIANGHAN - Henan Agricultural University|
|WANG, YONGZHEN - Xuchang University|
|ZHANG, MENG - Henan Agricultural University|
|LI, PENG - Henan Institute Of Science And Technology|
|LIU, MENG - China Institute Of Drug Control|
Submitted to: Journal of Virological Methods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/10/2019
Publication Date: 11/11/2019
Citation: Wang, D., Yu, J., Wang, Y., Zhang, M., Li, P., Liu, M., Liu, Y. 2019. Development of a real-time loop-mediated isothermal amplification (LAMP) assay and visual LAMP assay for detection of African Swine Fever Virus (ASFV). Journal of Virological Methods. 27. https://doi.org/10.1016/j.jviromet.2019.113775.
Interpretive Summary: African swine fever (ASF) is a fatal disease caused by a virus in domestic pigs. This disease is a serious concern in pig industry. Since no effective vaccines against African swine fever virus (ASFV) exist, there is a need to develop assays to detect the presence of ASFV. LAMP is a highly specific and sensitive DNA-based detection technology. We have developed Loop mediated isothermal amplification (LAMP) assays to detect ASFV. One lab-based assay has the potential to allow detection and quantitation of ASFV. A second LAMP assay was developed that is not quantitative, but results in a color change that can be seen by naked eye. Our results show that the developed LAMP assays are simple and cost-effective methods that can be used in clinical laboratories, in the field, or in less developed countries.
Technical Abstract: African swine fever (ASF) is a fatal disease caused by a virus in domestic pigs. In this study, a real-time loop-mediated isothermal amplification (LAMP) assay and visual LAMP assay were developed for the detection of African swine fever virus (ASFV). LAMP primers targeting the p10 gene of ASFV were designed, the LAMP reaction system was optimized with plasmid pUC57 containing the p10 gene sequence, and the specificities of the real-time LAMP and the visual assays were tested with the DNA or cDNA of pseudorabies virus (PRV), porcine circovirus type 2 (PCV2), classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine parvovirus (PPV) and ASFV, as well as the plasmid pUC57 containing the p10 gene sequence. The detection limits were determined using a serial dilution of plasmid pUC57 containing the p10 gene sequence. Our results showed that the LAMP assays could accurately and specifically detect ASFV with a detection limit of 30 copies per µl of pUC57 containing p10 gene sequence. In addition, the LAMP assays were evaluated using ASFV of various genotypes. Furthermore, the LAMP assays are comparable with the well-established real-time PCR assay. This study provides promising solutions for facilitating preliminary and cost-effective surveillance for prevention and control of ASFV.