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1 生物工程学报 Chinese Journal of Biotechnology DOI: /j.cjb 王经纬等 / 猪丁型冠状病毒荧光定量 RT-PCR 与 S1 蛋白间接 ELISA 检测方法的建立及应用 August 25, 2017, 33(8): Chin J Biotech, All rights reserved 新方法 1265 RT-PCR S1 ELISA 王经纬 1, 雷喜梅 1, 覃盼 1, 赵鹏伟 1, 王斌 1, 王逸雯 1, 李懿婷 1, 金颢蕊 1, 李龙 1 黄耀伟 ,,,,. RT-PCR S1 ELISA., 2017, 33(8): Wang JW, Lei XM, Qin P, et al. Development and application of real-time RT-PCR and S1 protein-based indirect ELISA for porcine deltacoronavirus. Chin J Biotech, 2017, 33(8): 摘要 : 最近, 新发现的猪丁型冠状病毒 (Porcine deltacoronavirus,pdcov) 可导致仔猪腹泻, 亟待建立有效准确的核酸及血清学检测方法并调查猪场的 PDCoV 感染情况本研究采用针对病毒 M 基因的探针荧光定量 RT-PCR 检测方法, 对年间收集的河南湖南浙江江西安徽河北黑龙江江苏山东和上海等 10 个省市收集的共 254 份腹泻仔猪小肠匀浆或粪便样本进行检测, 共检出 11 份 PDCoV 阳性样本, 总阳性率为 4.33% 采用在昆虫细胞表达纯化的 PDCoV 囊膜 S1 蛋白为包被抗原, 建立间接酶联免疫吸附试验 (ELISA) 方法, 检测年间收集的来自江苏湖南浙江等 7 个省份的 609 份具有腹泻症状的猪血清样品, 抗 PDCoV S1 抗体检出率为 44.17% (269/609) 上述建立的两种方法分别针对病毒的 RNA 或抗体对 PDCoV 进行特异性检测, 可以应用于 PDCoV 流行情况的监控 : 猪丁型冠状病毒, 实时荧光定量 RT-PCR, 间接 ELISA,S1 基因 Received: March 25, 2017; Accepted: June 9, 2017 Supported by: National Key Research and Development Program of China (No. 2016YFD ). Corresponding author: Yao-Wei Huang. Tel: ; yhuang@zju.edu.cn (No. 2016YFD )

2 1266 ISSN CN /Q Chin J Biotech August 25, 2017 Vol.33 No.8 Development and application of real-time RT-PCR and S1 protein-based indirect ELISA for porcine deltacoronavirus Jingwei Wang 1, Ximei Lei 1, Pan Qin 1, Pengwei Zhao 1, Bin Wang 1, Yiwen Wang 1, Yiting Li 1, Haorui Jin 1, Long Li 2, and Yao-Wei Huang 1 1 Institute of Preventive Veterinary Medicine, College of Animal Sciences, Zhejiang University, Hangzhou , Zhejiang, China 2 Hangzhou Belta-Biotechnology Co., Ltd., Hangzhou , Zhejiang, China Abstract: Porcine deltacoronavirus (PDCoV) has been recently recognized as an emerging viral pathogen that causes diarrhea in newborn piglets. A total of 254 small intestinal or fecal samples collected from 10 provinces including Henan, Hunan, Zhejiang, Jiangxi, Anhui, Hebei, Heilongjiang, Jiangsu, Shandong and Shanghai between 2014 and 2015, were screened by quantitative RT-PCR targeting the viral M gene. Eleven PDCoV positive samples were identified with a total positive rate of 4.33%. An indirect enzyme-linked immunosorbent assay (ELISA) was developed based on the recombinant S1 protein of PDCoV. This assay was used to test 609 serum samples of pigs with diarrhea symptoms collected from 10 provinces between 2015 and The positive rate of PDCoV antibody was 44.17% (269/609). The two methods can be used to monitor the PDCoV epidemiology in the levels of PDCoV specific RNA or antibody, helping better prevent and control PDCoV. Keywords: porcine deltacoronavirus, real-time RT-PCR, indirect ELISA, S1 gene (Deltacoronavirus) (Coronaviridae) (Coronavirinae) [1] 2009 (Porcine deltacoronavirus, PDCoV) 2014 PDCoV PDCoV HKU15 99% [2] 17 PDCoV 2014 PDCoV KOR/KNU14-04/2014 [3] Song PDCoV/CHJXNI2/2015 CH/Sichuan/S27/2012 [4-5] Chen CH/SXD1/2015 [6] Dong 3 [7] Dong CHN-AH PDCoV 2004 PDCoV [8-12] PDCoV [13-14] PDCoV 25.4 kb 5 3 ORF1ab S (S)

3 王经纬等 / 猪丁型冠状病毒荧光定量 RT-PCR 与 S1 蛋白间接 ELISA 检测方法的建立及应用 1267 (E) (M) (N) [15-16] PDCoV PDCoV PDCoV Marthaler ORF1 S M RT-PCR (qrt-pcr) 300 M PDCoV 30% [17] Song PDCoV N PCR PDCoV 33.71% (PEDV) 19.66% [4] Chen 6 64 RT-PCR PDCoV 23.4% [6] Thachil S (S1) ELISA 968 PDCoV 8.7% [18] Su N ELISA PDCoV [19] qrt-pcr PDCoV S1 ELISA PDCoV PDCoV 1 材料与方法 1.1 材料和试剂 10 TRIzol SuperScript Ⅲ Invitrogen Axygen Q5 NEB Thermo Fisher Scientific T7 High Efficiency Transcription Kit TransScript Probe One-Step qrt-pcr SuperMix IgG-Fc-HRP Abcam TMB ELISA ELISA ( PEDV TGEV ELISA ) 1.2 qrt-pcr 检测方法的建立 Marthaler M qrt-pcr SDCV-M-F 5 -ATCGACCACATGCGCTCAA-3 SDCV-M-R 5 -CAGCTCTTGCCCATGATCGT C-3 SDCV M-PROBE FAM-5 -CAC ACCAGTCGTTAAGCATGCGAGATT-3 -TAMRA PDCoV-TEMPLE-T7-F 5 -TAATACGACTCACTATAGGGATGTCTGACG CAGAAGAGTG-3 PDCoV-TEMPLE-T7-R cjb@im.ac.cn

4 1268 ISSN CN /Q Chin J Biotech August 25, 2017 Vol.33 No.8 5 -CTACGCTGCTGATT CCTGC-3 ( T7 ) 5 T7 PDCoV cdna M (1 988 bp) T7 High Efficiency Transcription Kit DNaseⅠ 15 min DNA TRIzol RNA RNA 10 TransScript Probe One-Step qrt-pcr SuperMix DNA C t qrt-pcr 1.3 间接 ELISA 检测方法建立 PDCoV S1 PCR S1 (aa1-571) S1 His tag pfast-bac DH10Bac Sf9 Sf9 P2 (10 8 TCID 50 /ml) Sf9 72 h Ni-NTA S1 SDS-PAGE Western blotting (ph 9.6) 2 S1 S µl PBST (0.01 mol/l PBS 0.05% Tween-20 ph 7.4) 3 PBST 5% 37 2 h PDCoV µl h PBST HRP IgG 100 µl/ min PBST µl TMB 15 min 50 µl/ 2 mol/l OD Western blotting [20] [18] PDCoV ELISA ELISA 609 PDCoV

5 王经纬等 / 猪丁型冠状病毒荧光定量 RT-PCR 与 S1 蛋白间接 ELISA 检测方法的建立及应用结果与分析 2.1 qrt- PCR 检测方法的建立 RNA ng/µl 10 6 qrt-pcr ( 1) R 2 = % ( 2) RNA ng/µl GE (Genome Equivalent) /µl PEDV TGEV ( ) qrt-pcr 0.20% 0.70% 0.30% 0.93% qrt-pcr PDCoV M (72 bp) qrt-pcr 图 1 Fig. 1 标准品的扩增曲线 The kinetic curve of the standard samples. 图 2 Fig. 2 标准品的标准曲线 The standard curve of the standard samples. 2.2 临床样本检测结果 PDCoV ( 7 4 ) 4.33% cjb@im.ac.cn

6 1270 ISSN CN /Q Chin J Biotech August 25, 2017 Vol.33 No.8 C t 22± GE/µL 11 PEDV 3 8 PEDV 72.73% 表 1 病料样本中 PDCoV RNA 检测结果统计 Table 1 Results of PDCoV RNA detected in specimen Samples Number of samples Positive samples Positive rate (%) Fecal Small intestine 表 2 病料样本来源 Table 2 The sources of the clinical specimens Area Number of samples Positive samples Positive rate (%) Henan Hunan Zhejiang Jiangxi Anhui Hebei Heilongjiang Jiangsu Shandong Shanghai Total 表 3 PDCoV RNA 阳性样本中 PEDV RNA 检测为阳性的样本信息 Table 3 Information about the PDCoV RNA positive specimen samples which are also positive of PEDV RNA Numbering Area Age Type Results of PEDV C t value Copies (GE/µL) JS-1 Jiangsu Nursery Fecal Positive JS-2 Jiangsu Nursery Fecal Positive JS-3 Jiangsu Nursery Fecal Positive JS-4 Jiangsu Nursery Fecal Positive JS-6 Jiangsu Nursery Fecal Positive JS-7 Jiangsu Nursery Fecal Positive JS-8 Jiangsu Nursery Fecal Positive Hunan Nursery Small intestine Positive Hunan Nursery Small intestine Negative Hunan Nursery Small intestine Negative Hunan Nursery Small intestine Negative PDCoV S1 蛋白表达与纯化 Sf9 P2 Sf9 72 h Ni + PDCoV S1 3B His Western blotting 3C PDCoV Western blotting ( ) ZS PDCoV Western blotting BCA 0.41 mg/ml

王经纬等 / 猪丁型冠状病毒荧光定量 RT-PCR 与 S1 蛋白间接 ELISA 检测方法的建立及应用 1271 图 3 重组 S1 蛋白纯化结果与 Western blotting 检测结果 Fig. 3 Purification and identification of the recombinant S1 protein expressed in insect cells.

7 王经纬等 / 猪丁型冠状病毒荧光定量 RT-PCR 与 S1 蛋白间接 ELISA 检测方法的建立及应用 1271 图 3 重组 S1 蛋白纯化结果与 Western blotting 检测结果 Fig. 3 Purification and identification of the recombinant S1 protein expressed in insect cells. (A) SDS-PAGE result of the purified recombinant S1 protein. (B) Detection of purified recombinant S1 using anti-his antibody by Western blotting. (C) Detection of purified recombinant S1 using PDCoV positive serum by Western blotting. M: protein marker ( kda); S1: purified recombinant S1 protein; Neg: the negative control (normal cell lysates); T: supernatants of infected-cell lysates; PS: the positive control (PDCoV positive serum); NS: the negative control (PDCoV negative serum); ZS: the negative control (The Bacmid without S1 sequences). 2.4 间接 ELISA 方法的建立及临床样本检测 1.25 ng/ HRP IgG ELISA 72 PDCoV x s x +3s=0.367 OD x +s=0.211 OD Thachil PDCoV S1 PEDV TGEV ELISA PEDV TGEV S1 OD S1 PEDV TGEV ELISA OD 450 =0.371 ELISA 1.46% 4.91% 2.01% 5.80% 10% ELISA S1 53 (R 2 =0.9415) ELISA ( ) ELISA PDCoV 44.17% 4 PDCoV 1 3 PDCoV 3 OD ± OD ±0.520 PDCoV PDCoV cjb@im.ac.cn

8 1272 ISSN CN /Q Chin J Biotech August 25, 2017 Vol.33 No.8 表 4 各年龄猪中抗 PDCoV 抗体检测结果统计 Table 4 Statistical data of anti-pdcov antibodies from pigs of different ages Samples Number of positive samples Number of samples Positive rate (%) Average value of OD 450 Gilts ±0.432 Sows ± week-olds ± week-olds ± week-olds ± week-olds ± week-olds ± week-olds ± week-olds ±0.301 Total ±0.421 PEDV TGEV PDCoV PDCoV PEDV TGEV PDCoV PEDV PDCoV PDCoV 图 4 不同年龄猪血清抗 PDCoV 抗体的分布情况 Fig. 4 Distribution of serum anti-pdcov antibodies obtained from pigs of different ages. The purified PDCoV S1 protein was used as the coating antigen and the indirect ELISA method was used to detect the OD 450 of 609 specific antibodies in porcine sera of different ages, the dotted line represents the cut-off value of ELISA. The graph was generated by GraphPad software. The solid line is the average of the OD 450 values for pigs of the same age. 3 讨论 qrt-pcr [21-22] PEDV TGEV RT-PCR qrt-pcr

9 王经纬等 / 猪丁型冠状病毒荧光定量 RT-PCR 与 S1 蛋白间接 ELISA 检测方法的建立及应用 1273 Chen [6] 23.4% Song [4] 33.71% PDCoV PDCoV 4.44% ( ) 1 PDCoV [5] PDCoV 72.73% PEDV PEDV PDCoV PDCoV PDCoV Su PDCoV N [19] ELISA PDCoV S S1 S [23] [24] S PDCoV S [25] ELISA PDCoV Ma PDCoV ELISA [26] PDCoV Thachil S1 ELISA S1 - Thachil S1 S1 Sf9 Sf9 His PDCoV 2 Western blotting PDCoV PDCoV Western blotting PDCoV S % Thachil 968 PDCoV 8.7% [18] Su % [19] PDCoV Hu PDCoV PDCoV [27] PDCoV PDCoV ELISA cjb@im.ac.cn

10 1274 ISSN CN /Q Chin J Biotech August 25, 2017 Vol.33 No.8 3 IgG PDCoV PDCoV PDCoV 5 PDCoV OD 450 PDCoV PDCoV PDCoV PDCoV PDCoV PDCoV REFERENCES [1] Woo PC, Lau SK, Lam CS, et al. Discovery of seven novel Mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus. J Virol, 2012, 86(7): [2] Wang L, Byrum B, Zhang Y. Detection and genetic characterization of deltacoronavirus in pigs, Ohio, USA, Emerg Infect Dis, 2014, 20(7): [3] Lee S, Lee C. Complete genome characterization of Korean porcine deltacoronavirus strain KOR/KNU14 04/2014. Genome Announc, 2014, 2(6): e [4] Song D, Zhou X, Peng Q, et al. Newly emerged porcine deltacoronavirus associated with diarrhoea in swine in China: identification, prevalence and full-length genome sequence analysis. Transbound Emerg Dis, 2015, 62(6): [5] Wang YW, Yue H, Fang WH, et al. Complete genome sequence of porcine deltacoronavirus strain CH/Sichuan/S27/2012 from mainland China. Genome Announc, 2015, 3(5): e [6] Chen FZ, Zhu YX, Wu MZ, et al. Full-Length genome characterization of Chinese porcine deltacoronavirus strain CH/SXD1/2015. Genome Announc, 2015, 3(5): e [7] Dong BQ, Liu W, Fan XH, et al. Detection of a novel and highly divergent coronavirus from Asian leopard cats and Chinese ferret badgers in Southern China. J Virol, 2007, 81(13): [8] Curry SM, Gibson KA, Burrough ER, et al. Nursery pig growth performance and tissue accretion modulation due to porcine epidemic diarrhea virus or porcine deltacoronavirus challenge. J Anim Sci, 2017, 95(1): [9] Fang PX, Fang LR, Dong N, et al. Research advances in the porcine deltacoronavirus. Chin J Virol, 2016, 32(2): [10] Janetanakit T, Lumyai M, Bunpapong N, et al. Porcine deltacoronavirus, Thailand, Emerg Infect Dis, 2016, 22(4): [11] Lee JH, Chung HC, Nguyen VG, et al. Detection and phylogenetic analysis of porcine deltacoronavirus in Korean swine farms, Transbound Emerg Dis, 2016, 63(3): [12] Lorsirigool A, Saeng-Chuto K, Temeeyasen G, et al. The first detection and full-length genome sequence of porcine deltacoronavirus isolated in Lao PDR. Arch Virol, 2016, 161(10):

11 王经纬等 / 猪丁型冠状病毒荧光定量 RT-PCR 与 S1 蛋白间接 ELISA 检测方法的建立及应用 1275 [13] Hu H, Jung K, Vlasova AN, et al. Isolation and characterization of porcine deltacoronavirus from pigs with diarrhea in the United States. J Clin Microbiol, 2015, 53(5): [14] Ma Y, Zhang Y, Liang X, et al. Origin, evolution, and virulence of porcine deltacoronaviruses in the United States. MBio, 2015, 6(2): e [15] Brian DA, Baric RS. Coronavirus genome structure and replication. Curr Top Microbiol Immunol, 2005, 287: [16] Wang YW, Huang YW. A newly discovered coronavirus genus-deltacoronavirus. Chem Life, 2016, 36(4): (in Chinese).,.., 2016, 36(4): [17] Marthaler D, Raymond L, Jiang Y, et al. Rapid detection, complete genome sequencing, and phylogenetic analysis of porcine deltacoronavirus. Emerg Infect Dis, 2014, 20(8): [18] Thachil A, Gerber PF, Xiao CT, et al. Development and application of an ELISA for the detection of porcine deltacoronavirus IgG antibodies. PLoS ONE, 2015, 10(4): e [19] Su MJ, Li CQ, Guo DH, et al. A recombinant nucleocapsid protein-based indirect enzyme-linked immunosorbent assay to detect antibodies against porcine deltacoronavirus. J Vet Med Sci, 2016, 78(4): [20] Huang YW, Harrall KK, Dryman BA, et al. Serological profile of torque teno sus virus species 1 (TTSuV1) in pigs and antigenic relationships between two TTSuV1 genotypes (1a and 1b), between two species (TTSuV1 and-2), and between porcine and human anelloviruses. J Virol, 2012, 86(19): [21] Narayanan K, Huang C, Lokugamage K, et al. Severe acute respiratory syndrome coronavirus nsp1 suppresses host gene expression, including that of type I interferon, in infected cells. J Virol, 2008, 82(9): [22] Neuman BW, Joseph JS, Saikatendu KS, et al. Proteomics analysis unravels the functional repertoire of coronavirus nonstructural protein 3. J Virol, 2008, 82(11): [23] Heald-Sargent T, Gallagher T. Ready, set, fuse! The coronavirus spike protein and acquisition of fusion competence. Viruses, 2012, 4(4): [24] Yang JH, Lü J, Wang YY, et al. Replication of murine coronavirus requires multiple cysteines in the endodomain of spike protein. Virology, 2012, 427(2): [25] Sun DB, Feng L, Shi HY, et al. Spike protein region (aa ) of porcine epidemic diarrhea virus is essential for induction of neutralizing antibodies. Acta Virol, 2007, 51(3): [26] Ma YM, Zhang Y, Liang XY, et al. Two-way antigenic cross-reactivity between porcine epidemic diarrhea virus and porcine deltacoronavirus. Vet Microbiol, 2016, 186: [27] Hu H, Jung K, Vlasova AN, et al. Experimental infection of gnotobiotic pigs with the cell-culture-adapted porcine deltacoronavirus strain OH-FD22. Arch Virol, 2016, 161(12): ( 本文责编陈宏宇 ) cjb@im.ac.cn

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<453A5CCAB3C6B7BFC6D1A73131C4EA3134C6DA5C3134C6DA5C3032B7D6CEF6> 227 ELISA 刘沙洲 1,2, 桑小雪 1, 欧阳华学 3, 雷绍荣 3, 白林含 1, * (1. 四川大学生命科学学院, 四川成都 610065;2. 成都市食品药品检测中心, 四川成都 610045; 3. 四川省农业科学院分析测试中心, 四川成都 610066) 目的 : 比较