Abstract：Abstract Porcine deltacoronavirus (PDCoV) is a new coronavirus that has been popular in China since the beginning of 2015. Development of a rapid and accurate detection method for the PDCoV is of great significance. A pair of primers were designed and synthesized according to the conserved region of the M gene sequence of PDCoV published in GenBank. The 260-bp fragment was successfully amplified and the recombinant plasmid of pMD18-T-PDCoV-M was constructed, and a SYBR GreenⅠreal-time RT-PCR was developed by optimization of the reacting conditions. The specificity, sensitivity and repeatability of the method were studied. The results demonstrated that the sensitivity of this assay was 54 copies/μL. In addition, the assay had good specificity for the PDCoV and had no cross-reaction with Transmissible gastroenteritis virus (TGEV), Pseudorabies virus (PRV), Porcine circovirus type 2 (PCV2), Porcine epidemic diarrhea virus (PEDV), and Porcine reproductive and respiratory syndrome virus (PRRSV). This SYBR Green Ⅰreal-time RT-PCR showed good repeatability, and the variations in intra- and inter-assays were both less than 1%. 198 clinical samples were tested for PDCoV by using this established method, and the positive rate was 20.71% (41/198). The established SYBR GreenⅠRT-PCR method for PDCoV provides a rapid and accurate diagnosis for PDCoV and provides a new technical means for the further study of the disease.
Hu H, Jung K, Vlasova AN, et al. Isolation and Characterization of Porcine Deltacoronavirus from Pigs with Diarrhea in the United States [J]. J Clin Microbiol, 2015, 21(4): 1537-1548.
Jung K, Hu H, Eyerly B, et al. Pathogenicity of 2 Porcine Deltacoronavirus Strains in Gnotobiotic Pigss [J]. Emerg. Infect. Dis, 2015, 21(4): 650-654.
Wang L, Byrum B, Zhang Y. Detection and genetic characterization of deltacoronavirus in pigs, Ohio, USA, 2014.[J]. Emerg Infect Dis, 2014, 20: 1227-1230.
Marthaler D, Raymond L, Jiang Y, et al. Rapid detection, complete genome sequencing, and phylogenetic analysis of porcine deltacoronavirus [J]. Emerg Infect Dis, 2014, 20: 1347-1350.
Li G, Chen Q, Harmon KM, et al. Full-Length Genome Sequence of Porcine Deltacoronavirus Strain USA/IA/2014/8734 [J]. Genome Announc , 2014, 2(2): e00278-14.
Wang L, Byrum B, Zhang Y. Porcine coronavirus HKU15 detected in 9 US states, 2014. [J]. Emerg Infect Dis, 2014, 20(9): 1594-1595.
Marthaler D, Jiang Y, Collins J, et al. Complete Genome Sequence of Strain SDCV/USA/Illinois121/2014, a Porcine Deltacoronavirus from the United States [J].Genome Announc , 2014, 2(2): e00218-14.
Lee S, Lee C. Complete Genome Characterization of Korean Porcine Deltacoronavirus Strain KOR/KNU14-04/2014 [J]. Genome Announcements, 2014, 2(6): 1-2.
Janetanakit T, Lumyai M, Bunpapong N, et al. Porcine Deltacoronavirus, Thailand, 2015[J]. Emerg Infect Dis, 2016, 22(4): 757-759.
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].J Virol, 2012, 86: 3995-4008.
逢凤娇, 俞正玉, 何孔旺, 等. 猪Deltacoronavirus RT-PCR检测方法的建立及其应用[J]. 中国预防兽医学报, 2015, 37(9): 683-686. （Feng F J|, Yu Z Y, He K W, et al. Development and application of a RT-PCR assay for detection of porcine Deltacoronavirus [J]. Chinese Journal of Preventive Veterinary Medicine, 2015, 37(9): 683-686. )
张帆帆, 宋德平, 周信荣, 等. 新现猪Delta冠状病毒RT-PCR检测方法的建立及其应用[J]. 中国农业科学, 2016, 49(7): 1408-1416.（Zhang F F, Song D P, Zhou X R,et al. Establishment and Application of a RT-PCR Assay for Detection of Newly Emerged Porcine Deltacoronavirus[J]. AgriculturalSciencesinChina, 2016, 49(7): 1408-1416. ）
刘玲玲，曹贝贝，张利卫，等. 新发猪Delta冠状病毒和猪传染性胃肠炎病毒双重RT-PCR检测方法的建立及初步应用[J].农业生物技术学报，2016，24（12）；1955-1963. （Liu L L,Cao B B,Zhang L W,et al.Establishment and Preliminary Application of Duplex Reverse Transcription-PCR for Detection of Emerging Porcine Deltacoronavirus and Porcine Transmissible Gastroenteritis Virus[J]，Journal of Agricultural Biotechnology, 2016，24（12）；1955-1963.
Song D, Zhou X, Chen O, et al. Newly Emerged Porcine Deltacoronavirus Associated With Diarrhoea in Swine in China: Identification, Prevalence and Full-Length Genome Sequence Analysis [J]. Transboundary and Emerging Diseases, 2015, 62: 575-580.
Ma Y M, Zhang Y, Liang X Y, et al. Origin, evolution, and virulence of porcine deltacoronaviruse in the United States[J]. MBio, 2015, 6(2): e00064.
