猪急性腹泻综合征冠状病毒SYBR Green荧光定量PCR检测方法的建立及应用

doi:10.11843/j.issn.0366-6964.2021.010.019

Abstract

Abstract: To establish a rapid, sensitive and specific detection method for swine acute diarrhea syndrome coronavirus (SADS-CoV), the conserved region of the SADS-CoV N gene was amplified and cloned into pMD18-T vector. The recombinant plasmid pMD18-T-SADS-qN was used as the positive plasmid standard to establish a SYBR Green real-time PCR. The results showed that the method showed a good linear relationship when the template was among in 3.31×10¹-3.31×10⁷ copies·μL^-1, with a correlation coefficient (R²) of 0.997, and a slope of -3.318. The detection of SADS-CoV was specific, and that porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine delta coronavirus (PDCoV) and porcine reproductive and respiratory syndrome virus (PRRSV) were all tested negative by this method. This detection technology was sensitive and repeatable with the limited detection contend of the standard plasmid being 3.31×10¹ copies·μL^-1 and the intra-group and inter-group coefficients of variation being less than 1%. This method was used to detect the replication of IPI-2I and IPEC-J2 cells infected with SADS-CoV at different time points and different doses. The results showed that the viral load of IPI-2I and IPEC-J2 cells infected with SADS-CoV stayed at a low level at 2 h postinfection; after at 12-36 h postinfection increased rapidly; and at 36 h postinfection the growth rate slowed down, virus RNA maintained at a high level. The results of infection of cells with 0, 0.1, 1 MOI SADS-CoV showed that the mRAN transcription level of the virus showed a dose-dependent increase. When infected with SADS-CoV at an MOI of 1, the viral load in IPI-2I and IPEC-J2 cells was 10^6.7 and 10^5.3 copies·mL^-1, respectively. The established method was further used to detect the clinical samples of piglets that infection with SADS-CoV by oral route. High levels of viral RNA copies were detected in jejunum and ileum, indicating that the virus mainly colonized in jejunum and ileum. In conclusion, the SYBR Green real-time PCR method established in this study can detect SADS-CoV sensitively and specifically, and provide a reliable detection method for the diagnosis of SADS-CoV and virus related basic researches.

Key words: swine acute diarrhea syndrome coronavirus (SADS-CoV), N gene, real-time PCR, detection

CLC Number:

S852.659.6

ZHANG Jiyu, HAN Yuru, SHI Hongyan, CHEN Jianfei, ZHANG Xin, LIU Jianbo, ZHANG Liaoyuan, FENG Shufeng, FENG Tingshuai, JI Zhaoyang, SHI Da, FENG Li. Establishment and Application of SYBR Green Real-time PCR for Swine Acute Diarrhea Syndrome Coronavirus[J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(10): 2887-2894.

References 15

[1]	ZHOU P, FAN H, LAN T, et al. Fatal swine acute diarrhoea syndrome caused by an HKU2-related coronavirus of bat origin[J]. Nature, 2018, 556(7700):255-258.
[2]	PAN Y F, TIAN X Y, QIN P, et al. Discovery of a novel swine enteric alphacoronavirus (SeACoV) in southern China[J]. Vet Microbiol, 2017, 211:15-21.
[3]	WANG H N, CONG F, ZENG F W, et al. Development of a real time reverse transcription loop-mediated isothermal amplification method (RT-LAMP) for detection of a novel swine acute diarrhea syndrome coronavirus (SADS-CoV)[J]. J Virol Methods, 2018, 260:45-48.
[4]	MACKAY I M, ARDEN K E, NITSCHE A. Real-time PCR in virology[J]. Nucleic Acids Res, 2002, 30(6):1292-1305.
[5]	GINZINGER D G. Gene quantification using real-time quantitative PCR:an emerging technology hits the mainstream[J]. Exp Hematol, 2002, 30(6):503-512.
[6]	GONG L, LI J, ZHOU Q F, et al. A new Bat-HKU2-like coronavirus in Swine, China, 2017[J]. Emerg Infect Dis, 2017, 23(9):1607-1609.
[7]	MUNGMUNGPUNTIPANTIP R, WIWANITKIT V. RT-PCR Tests for COVID-19:observation on use and cost[J]. Clin Lab, 2020, 66.
[8]	LIU Z Y, LI W, XU M, et al. Development of an in-house multiplex nested RT-PCR method for detecting acute HIV-1 infection in high risk populations[J]. Curr HIV Res, 2015, 13(4):315-324.
[9]	HU H, JUNG K, WANG Q H, et al. Development of a one-step RT-PCR assay for detection of pancoronaviruses (α-, β-, γ-, and δ-coronaviruses) using newly designed degenerate primers for porcine and avian fecal samples[J]. J Virol Methods, 2018, 256:116-122.
[10]	ZHOU L, SUN Y, WU J L, et al. Development of a TaqMan-based real-time RT-PCR assay for the detection of SADS-CoV associated with severe diarrhea disease in pigs[J]. J Virol Methods, 2018, 255:66-70.
[11]	MA L, ZENG F W, CONG F, et al. Development of a SYBR green-based real-time RT-PCR assay for rapid detection of the emerging swine acute diarrhea syndrome coronavirus[J]. J Virol Methods, 2019, 265:66-70.
[12]	EDWARDS C E, YOUNT B L, GRAHAM R L, et al. Swine acute diarrhea syndrome coronavirus replication in primary human cells reveals potential susceptibility to infection[J]. Proc Natl Acad Sci U S A, 2020, 117(43):26915-26925.
[13]	YANG Y L, QIN P, WANG B, et al. Broad cross-species infection of cultured cells by bat HKU2-related swine acute diarrhea syndrome coronavirus and identification of its replication in murine dendritic cells in vivo highlight its potential for diverse interspecies transmission[J]. J Virol, 2019, 93(24):e01448-19.
[14]	WANG X L, FANG L R, LIU S D, et al. Susceptibility of porcine IPI-2I intestinal epithelial cells to infection with swine enteric coronaviruses[J]. Vet Microbiol, 2019, 233:21-27.
[15]	KROPINSKI A M. Practical advice on the one-step growth curve[M]//CLOKIE M, KROPINSKI A, LAVIGNE R, et al. Bacteriophages. Methods in Molecular Biology. New York, NY:Humana Press, 2018, 1681:41-47.

Establishment and Application of SYBR Green Real-time PCR for Swine Acute Diarrhea Syndrome Coronavirus

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

References 15

Related Articles 15

Recommended Articles

Metrics

Comments

[1]	CHONG Qian, ZHAO Xuehui, CAO Qing, XUE Huiwen, GOU Huitian. Research Progress of Applied Research on Listeria monocytogenes Phage [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(5): 1819-1826.
[2]	ZHANG Shaohua, WANG Shuai, ZOU Yang, LIU Zhongli, CAI Xuepeng. Advances in Detection Approaches for Ovine Haemonchosis [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(4): 1499-1510.
[3]	YANG Heng, LI Zhanhong, SONG Zi'ang, GAO Lin, LI Zhuoran, LIAO Defang, XIAO Lei, LI Huachun. Development and Application of Serotyping Real-time Quantitative RT-PCR Methods for Palyam Virus [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(1): 395-400.
[4]	JIANG Lingling, NIU Xiaoxia, LIU Qiang, ZHANG Gang, WANG Pu, LI Yong. Analysis of Infection Status of Beef Cattle Diarrhea-related Virus in Ningxia [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(9): 3863-3871.
[5]	LIU Hua, YIN Dongdong, SHAO Ying, SONG Xiangjun, WANG Zhenyu, PAN Xiaocheng, TU Jian, HE Changsheng, ZHU Liangqiang, QI Kezong. Detection of Porcine Epidemic Diarrhea Virus by Recombinase Aided Amplification Combined with CRISPR/Cas13a [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(9): 3991-3997.
[6]	HU Xiangyun, CAO Yanhong, LÜ Lingyan, LIU Zheng, HUANG Facai, WU Zhuyue, XIAO Zhengzhong. Nanobodies and Their Research Status in Veterinary Field [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(8): 3164-3172.
[7]	ZHANG Ping, ZHUANG Linlin, ZHANG Di, DONG Yongyi, SHENG Zhongwei, WANG Chengming, XU Bu, DOU Xinhong, GONG Jiansen. Research Progress on Molecular Detection Methods of Salmonella [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(8): 3217-3229.
[8]	CHEN Hongjian, CAO Yan, FAN Jie, GAN Rongxuan, SONG Wenbo, YU Shengwei, YANG Ting, ZHAO Yanxia, WEI Chunyan, XIE Rui, HUA Lin, PENG Zhong, WU Bin. Prevalence and Phylogenetic Analysis of Pseudorabies Virus within Pig Slaughterhouses in Hubei Province of China during 2020-2022 [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(7): 2972-2981.
[9]	LI Zhaoyan, GAO Jiang, GUO Shihui, ZHAO Ruqian, MA Wenqiang. Advances in Detection Methods and Control Solutions for Feline Allergens [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(6): 2272-2279.
[10]	CAO Liyan, KONG Xiangyu, LI Xiangtong, SUO Xuepeng, DUAN Yueyue, YUAN Cong, SHI Lei, ZHANG Yu, MA Guoxiang, ZHENG Haixue, WANG Qi. Preparation and Sequence Analysis of Monoclonal Antibody against the Nucleocapsid Protein of Porcine Acute Diarrhea Syndrome Coronavirus [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(6): 2487-2497.
[11]	CHEN Yang, MENG Linchun, GUO Mengjiao, ZHANG Chengcheng, BO Zongyi, CHU Dianfeng, CAO Yongzhong, WU Yantao, ZHANG Xiaorong. Establishment and Preliminary Application of Indirect ELISA Method and HI Test for Detection of Mycoplasma Gallisepticum Antibody [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(5): 2062-2072.
[12]	Lü Qiao, ZHAO Zhongyi, YIN Dewei, LIU Yumeng, WANG Wei, ZHENG Min, HU Shiyue, ZHAO Chenchen, ZHANG Xinyu, LEI Xiaoxiao, LU Jingyi, SUN Wenchao, LAN Tian. Establishment and Preliminary Application of a Real-time RT-RAA for Detection of Transmissible Gastroenteritis Virus [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(5): 2208-2214.
[13]	ABI-kehamo, TANG Cheng, YANG Chen, YANG Falong. Research Advances in Kobuvirus [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(3): 900-913.
[14]	HE Shufan, ZOU Yuantong, HUANG Zhilan, LI Qian, JIANGCUO Wengxi, YUE Hua, TANG Cheng, LIU Jie. Detection of Bovine Adenovirus Type 3 Infection Status and Its Fiber Shaft Gene [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(3): 1333-1340.
[15]	GAO Pingping, FU Jinyu, WANG Liyang, SHI Shuobo, ZHANG Yueping, ZHANG Di. Rapid RPA-CRISPR/Cas12a Detection Platform for Dermatophytes in Dogs and Cats [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(11): 4702-4711.