PMID- 35733636 OWN - NLM STAT- PubMed-not-MEDLINE LR - 20220716 IS - 2297-1769 (Print) IS - 2297-1769 (Electronic) IS - 2297-1769 (Linking) VI - 9 DP - 2022 TI - Development and Application of a Duplex Droplet Digital Polymerase Chain Reaction Assay for Detection and Differentiation of EP402R-Deleted and Wild-Type African Swine Fever Virus. PG - 905706 LID - 10.3389/fvets.2022.905706 [doi] LID - 905706 AB - African swine fever (ASF) is a highly fatal porcine disease caused by the African swine fever virus (ASFV), and resulting in huge economic losses across the globe. ASF has been raging in China for 3 years, and recently EP402R-deleted ASFV strains emerged, showing sub-acute or chronic symptoms in pigs and providing novel difficulties to monitor and control the disease as EP402R-deleted strains possess no hemadsorption (HAD) ability. In addition, the gene deletion virus with low viral load is prone to results retest or false negative due to the high cycle threshold (Ct) value under the current real-time polymerase chain reaction (PCR) detection method. Thus, a new method is needed to detect and distinguish wild strains and gene-deleted viruses. In this study, a duplex droplet digital polymerase chain reaction (ddPCR) assay based on the ASFV B646L and EP402R genes was established and showed good linearity (R(2) > 0.99). The limit of detection for duplex ddPCR was 52 copies per reaction and 8.6 copies per reaction for B646L and EP402R, respectively. No cross-reaction with other porcine viruses [classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine epidemic diarrhea virus (PEDV), porcine parvovirus (PPV), Japanese encephalitis virus (JEV), and porcine circovirus type 2 (PCV2)] was identified by this assay. In addition, 44 ASFV-suspicious clinical samples as well as EP402R-deleted ASFV were tested in parallel by duplex real-time PCR and ddPCR, indicative of a higher sensitivity which belonged to the duplex ddPCR assay. In summary, this is the first time that duplex ddPCR assay has been successfully developed to provide an efficient method to detect and differentiate ASFV wild-type and gene-deleted strains. CI - Copyright (c) 2022 Zhu, Jian, Huang, Gao, Gao, Chen, Zhang, Liao and Qi. FAU - Zhu, Junhai AU - Zhu J AD - College of Veterinary Medicine, South China Agricultural University, Guangzhou, China. AD - African Swine Fever Regional Laboratory of China (Guangzhou), Guangzhou, China. AD - Key Laboratory of Zoonoses, Ministry of Agriculture and Rural Affairs of the People's Republic of China, Guangzhou, China. AD - National and Regional Joint Engineering Laboratory for Medicament of Zoonoses Prevention and Control, National Development and Reform Commission of the People's Republic of China, Guangzhou, China. AD - Key Laboratory of Zoonoses Prevention and Control of Guangdong, Guangzhou, China. FAU - Jian, Weijun AU - Jian W AD - College of Veterinary Medicine, South China Agricultural University, Guangzhou, China. AD - African Swine Fever Regional Laboratory of China (Guangzhou), Guangzhou, China. AD - Key Laboratory of Zoonoses, Ministry of Agriculture and Rural Affairs of the People's Republic of China, Guangzhou, China. AD - National and Regional Joint Engineering Laboratory for Medicament of Zoonoses Prevention and Control, National Development and Reform Commission of the People's Republic of China, Guangzhou, China. FAU - Huang, Yifan AU - Huang Y AD - College of Veterinary Medicine, South China Agricultural University, Guangzhou, China. AD - African Swine Fever Regional Laboratory of China (Guangzhou), Guangzhou, China. AD - Key Laboratory of Zoonoses, Ministry of Agriculture and Rural Affairs of the People's Republic of China, Guangzhou, China. AD - National and Regional Joint Engineering Laboratory for Medicament of Zoonoses Prevention and Control, National Development and Reform Commission of the People's Republic of China, Guangzhou, China. FAU - Gao, Qi AU - Gao Q AD - College of Veterinary Medicine, South China Agricultural University, Guangzhou, China. AD - African Swine Fever Regional Laboratory of China (Guangzhou), Guangzhou, China. AD - Key Laboratory of Zoonoses, Ministry of Agriculture and Rural Affairs of the People's Republic of China, Guangzhou, China. AD - National and Regional Joint Engineering Laboratory for Medicament of Zoonoses Prevention and Control, National Development and Reform Commission of the People's Republic of China, Guangzhou, China. FAU - Gao, Fei AU - Gao F AD - College of Veterinary Medicine, South China Agricultural University, Guangzhou, China. AD - African Swine Fever Regional Laboratory of China (Guangzhou), Guangzhou, China. AD - Key Laboratory of Zoonoses, Ministry of Agriculture and Rural Affairs of the People's Republic of China, Guangzhou, China. AD - National and Regional Joint Engineering Laboratory for Medicament of Zoonoses Prevention and Control, National Development and Reform Commission of the People's Republic of China, Guangzhou, China. FAU - Chen, Huahan AU - Chen H AD - College of Veterinary Medicine, South China Agricultural University, Guangzhou, China. AD - African Swine Fever Regional Laboratory of China (Guangzhou), Guangzhou, China. AD - Key Laboratory of Zoonoses, Ministry of Agriculture and Rural Affairs of the People's Republic of China, Guangzhou, China. AD - National and Regional Joint Engineering Laboratory for Medicament of Zoonoses Prevention and Control, National Development and Reform Commission of the People's Republic of China, Guangzhou, China. FAU - Zhang, Guihong AU - Zhang G AD - College of Veterinary Medicine, South China Agricultural University, Guangzhou, China. AD - African Swine Fever Regional Laboratory of China (Guangzhou), Guangzhou, China. AD - Key Laboratory of Zoonoses, Ministry of Agriculture and Rural Affairs of the People's Republic of China, Guangzhou, China. AD - National and Regional Joint Engineering Laboratory for Medicament of Zoonoses Prevention and Control, National Development and Reform Commission of the People's Republic of China, Guangzhou, China. FAU - Liao, Ming AU - Liao M AD - College of Veterinary Medicine, South China Agricultural University, Guangzhou, China. AD - African Swine Fever Regional Laboratory of China (Guangzhou), Guangzhou, China. AD - Key Laboratory of Zoonoses, Ministry of Agriculture and Rural Affairs of the People's Republic of China, Guangzhou, China. AD - National and Regional Joint Engineering Laboratory for Medicament of Zoonoses Prevention and Control, National Development and Reform Commission of the People's Republic of China, Guangzhou, China. AD - Key Laboratory of Zoonoses Prevention and Control of Guangdong, Guangzhou, China. FAU - Qi, Wenbao AU - Qi W AD - College of Veterinary Medicine, South China Agricultural University, Guangzhou, China. AD - African Swine Fever Regional Laboratory of China (Guangzhou), Guangzhou, China. AD - Key Laboratory of Zoonoses, Ministry of Agriculture and Rural Affairs of the People's Republic of China, Guangzhou, China. AD - National and Regional Joint Engineering Laboratory for Medicament of Zoonoses Prevention and Control, National Development and Reform Commission of the People's Republic of China, Guangzhou, China. AD - Key Laboratory of Zoonoses Prevention and Control of Guangdong, Guangzhou, China. LA - eng PT - Journal Article DEP - 20220606 PL - Switzerland TA - Front Vet Sci JT - Frontiers in veterinary science JID - 101666658 PMC - PMC9207387 OTO - NOTNLM OT - African swine fever virus OT - droplet digital polymerase chain reaction OT - gene-deleted strain differentiation OT - laboratory diagnosis OT - sensitive detection COIS- The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. EDAT- 2022/06/24 06:00 MHDA- 2022/06/24 06:01 PMCR- 2022/01/01 CRDT- 2022/06/23 02:26 PHST- 2022/03/27 00:00 [received] PHST- 2022/05/04 00:00 [accepted] PHST- 2022/06/23 02:26 [entrez] PHST- 2022/06/24 06:00 [pubmed] PHST- 2022/06/24 06:01 [medline] PHST- 2022/01/01 00:00 [pmc-release] AID - 10.3389/fvets.2022.905706 [doi] PST - epublish SO - Front Vet Sci. 2022 Jun 6;9:905706. doi: 10.3389/fvets.2022.905706. eCollection 2022.