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.

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

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