一种快速检测非洲猪瘟病毒I177L基因缺失毒株的荧光定量PCR方法

doi:10.11843/j.issn.0366-6964.2023.06.030

Acta Veterinaria et Zootechnica Sinica ›› 2023, Vol. 54 ›› Issue (6): 2521-2527.doi: 10.11843/j.issn.0366-6964.2023.06.030

• PREVENTIVE VETERINARY MEDICINE • Previous Articles Next Articles

A Real-time PCR Method for the Rapid Detection of African Swine Fever Virus I177L Gene Deletion Strain

QIU Yingwu^1,2, CHANG Hao^3,4, PENG Jie^1,4, YI Heyou^1,3, WANG Qiumei^1,4, GUO Yanchen^1,3, WU Qianwen^1,3, CAO Xuezhen², LIN Limiao², LI Wei², ZHOU Qingfeng², ZHANG Guihong^1,3,4, LI Qunhui^2*, GONG Lang^1,3,5*

1. Research Center of African Swine Fever Prevention and Control Technology, College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, China;
2. Guangdong Provincial Key Laboratory of Livestock and Poultry Health and Environmental Control, Xinxing 527400, China;
3. National African Swine Fever Regional Laboratory(Guangzhou), Guangzhou 510642, China;
4. Guangdong Provincial Key Laboratory for Prevention and Control of Zoonoses, Guangzhou 510642, China;
5. Guangdong Provincial Laboratory of Modern Agricultural Science and Technology, Maoming Branch Center, Maoming 525000, China

Received:2022-11-14 Online:2023-06-23 Published:2023-06-16

Abstract

Abstract: The aim of this study was to establish a specific, sensitive and rapid real-time fluorescence quantitative PCR (FQ-PCR) method for the detection of ASFV-G-ΔI177L. Specific primers/probes were designed for the conserved region of ASFV I117L gene to improve the sensitivity of the detection method and verify the repeatability and specificity of the detection method by optimizing the reaction conditions and procedures. A total of 170 clinical samples were tested and compared with OIE recommended testing methods. The results showed that the specific primer/probe pair based on ASFV I117L gene could amplify 2.2×10¹-2.2×10¹⁰ copies·μL^-1 pMD18-I177L standard plasmid, and the established standard curve R² reached 0.995 7. The minimum detection template concentration was 2.2×10¹copies·μL^-1. The amplification reaction was performed by one-step method, which took 35 min, and the coefficient of variation within and between batches was less than 1.8%. Specific amplification of ASFV I177L gene was detected, but no positive amplification was observed for nucleic acid samples of other 5 common porcine viruses and enzyme-free water. This method was used to detect 170 clinical samples, and it was in good agreement with the method recommended by OIE. This study successfully established a specific, sensitive and rapid ASFV-G-ΔI177L detection method, which provides a good technical support for the clinical monitoring and diagnosis of African swine fever virus.

Key words: ASF, ASFV, TaqMan FQ-PCR, molecular diagnosis

CLC Number:

S852.659.1

QIU Yingwu, CHANG Hao, PENG Jie, YI Heyou, WANG Qiumei, GUO Yanchen, WU Qianwen, CAO Xuezhen, LIN Limiao, LI Wei, ZHOU Qingfeng, ZHANG Guihong, LI Qunhui, GONG Lang. A Real-time PCR Method for the Rapid Detection of African Swine Fever Virus I177L Gene Deletion Strain[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(6): 2521-2527.

References 14

[1]	WU K K, LIU J M, WANG L X, et al. Current state of global African Swine fever vaccine development under the prevalence and transmission of ASF in China[J]. Vaccines (Basel), 2020, 8(3):531.
[2]	FAN Y Q, CHEN W Y, JIANG C G, et al. Host Responses to Live-Attenuated ASFV (HLJ/18-7GD)[J]. Viruses, 2022, 14(9):2003.
[3]	CHATHURANGA K, LEE J S. African swine fever virus (ASFV):immunity and vaccine development[J]. Vaccines (Basel), 2023, 11(2):199.
[4]	WANG Z Y, AI Q Y, HUANG S L, et al. Immune escape mechanism and vaccine research progress of African swine fever virus[J]. Vaccines (Basel), 2022, 10(3):344.
[5]	TRAN X H, PHUONG L T T, HUY N Q, et al. Evaluation of the safety profile of the ASFV vaccine candidate ASFV-G-ΔI177L[J]. Viruses, 2022, 14(5):896.
[6]	URBANO A C, FERREIRA F. African swine fever control and prevention:an update on vaccine development[J]. Emerg Microbes Infect, 2022, 11(1):2021-2033.
[7]	陈孝军, 马俊, 陈熊男, 等. 1株非洲猪瘟病毒p30蛋白单克隆抗体识别的B细胞抗原表位的鉴定[J]. 畜牧兽医学报, 2022, 53(7): 2239-2251.CHEN X J, MA J, CHEN X N, et al. Identification of the B cell epitopes recognized by a monoclonal antibody against the p30 protein of African swine fever virus[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(7): 2239-2251. (in Chinese)
[8]	HUANG Z, XU Z Y, CAO H X, et al. A Triplex PCR method for distinguishing the wild-type African swine fever virus from the deletion strains by detecting the gene insertion[J]. Front Vet Sci, 2022, 9:921907.
[9]	CAO S N, LU H P, WU Z, et al. A duplex fluorescent quantitative PCR assay to distinguish the genotype I and II strains of African swine fever virus in Chinese epidemic strains[J]. Front Vet Sci, 2022, 9:998874.
[10]	WANG G G, XIE M J, WU W, et al. Structures and functional diversities of ASFV proteins[J]. Viruses, 2021, 13(11):2124.
[11]	LI Z Y, CHEN W X, QIU Z L, et al. African swine fever virus:a review[J]. Life (Basel), 2022, 12(8):1255.
[12]	王涛, 孙元, 罗玉子, 等. 非洲猪瘟防控及疫苗研发:挑战与对策[J]. 生物工程学报, 2018, 34(12): 1931-1942.WANG T, SUN Y, LUO Y Z, et al. Prevention, control and vaccine development of African swine fever:challenges and countermeasures[J]. Chinese Journal of Biotechnology, 2018, 34(12):1931-1942.(in Chinese)
[13]	BORCA M V, RAI A, RAMIREZ-MEDINA E, et al. A cell culture-adapted vaccine virus against the current African swine fever virus pandemic strain[J]. J Virol, 2021, 95(14):e0012321.
[14]	曾繁聪, 姜含雨, 辛宁, 等. 基于非洲猪瘟病毒I177L和p72基因的双重TaqMan荧光定量PCR鉴别检测方法的建立[J]. 中国预防兽医学报, 2022, 44(9):952-957.ZENG F C, JIANG H Y, XIN N, et al. The development of a duplex TaqMan real-time PCR assay targeting the African swine fever virus I177L and p72 genes[J]. Chinese Journal of Preventive Veterinary Medicine, 2022, 44(9):952-957. (in Chinese)

A Real-time PCR Method for the Rapid Detection of African Swine Fever Virus I177L Gene Deletion Strain

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