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

畜牧兽医学报 ›› 2021, Vol. 52 ›› Issue (10): 2887-2894.doi: 10.11843/j.issn.0366-6964.2021.010.019

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

张记宇, 韩郁茹, 时洪艳, 陈建飞, 张鑫, 刘建波, 张燎原, 冯书风, 冯廷帅, 季朝阳, 石达*, 冯力*

中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室猪消化道传染病创新团队, 哈尔滨 150069

收稿日期:2021-01-21 出版日期:2021-10-23 发布日期:2021-10-27
通讯作者: 石达,主要从事兽医微生物及其分子生物学研究,E-mail:shida@caas.cn;冯力,主要从事兽医微生物及其分子生物学研究,E-mail:fengli@caas.cn
作者简介:张记宇(1996-),女,山西柳林人,硕士生,主要从事动物病毒及分子生物学研究,E-mail:zhangjiyu0429@163.com
基金资助:
黑龙江省自然科学基金研究团队项目（TD2020C002）；黑龙江省自然科学基金（C2018066）

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

ZHANG Jiyu, HAN Yuru, SHI Hongyan, CHEN Jianfei, ZHANG Xin, LIU Jianbo, ZHANG Liaoyuan, FENG Shufeng, FENG Tingshuai, JI Zhaoyang, SHI Da*, FENG Li*

Swine Digestive System Infectious Diseases Division, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150069, China

Received:2021-01-21 Online:2021-10-23 Published:2021-10-27

摘要/Abstract

摘要： 为建立快速、灵敏且特异的检测猪急性腹泻综合征冠状病毒（swine acute diarrhea syndrome coronavirus，SADS-CoV）检测方法，本试验扩增SADS-CoV N基因保守区域将其克隆至pMD18-T载体。所构建的重组质粒pMD18-T-SADS-qN作为阳性质粒标准品，以其为模板建立一种SYBR Green荧光定量PCR检测方法。结果显示，所建立方法在3.31×10¹~3.31×10⁷拷贝·μL^-1模板量时，呈良好的线性关系，相关系数（R²）为0.997，斜率为-3.318。该方法特异性检测SADS-CoV；而猪流行性腹泻病毒（PEDV）、猪传染性胃肠炎病毒（TGEV）、猪德尔塔冠状病毒（PDCoV）和猪繁殖与呼吸综合征病毒（PRRSV）检测结果均为阴性。所构建的标准品检测灵敏度下限可以达到3.31×10¹拷贝·μL^-1，组内和组间变异系数均小于1%，表明其具有良好的灵敏性和重复性。用该方法检测SADS-CoV感染IPI-2I和IPEC-J2细胞后不同时间点和不同接毒剂量的复制情况，结果显示，SADS-CoV感染细胞后2 h病毒含量较低，在12~36 h病毒含量迅速增长，36 h后增长速度减缓且病毒含量维持在较高水平。分别用0、0.1、1 MOI SADS-CoV感染细胞结果显示病毒的mRAN转录水平呈现剂量依赖性增加，当MOI为1时，IPI-2I和IPEC-J2细胞病毒含量分别为10^6.7、10^5.3拷贝·mL^-1。进一步利用所建立的方法对经口服攻毒SADS-CoV仔猪的临床样本进行检测，结果发现病毒在空肠回肠含量较高，表明病毒主要定殖于空肠和回肠。综上表明，本研究建立SYBR Green荧光定量PCR检测方法能灵敏特异地检测SADS-CoV，为SADS-CoV的诊断和病毒相关基础研究提供可靠的检测手段。

关键词: 猪急性腹泻综合征病毒, N基因, 荧光定量PCR, 检测

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

中图分类号:

S852.659.6

张记宇, 韩郁茹, 时洪艳, 陈建飞, 张鑫, 刘建波, 张燎原, 冯书风, 冯廷帅, 季朝阳, 石达, 冯力. 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[J]. 畜牧兽医学报, 2021, 52(10): 2887-2894.

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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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

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

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