Establishment and Preliminary Application of Duplex RT-PCR for Detection of Emerging Porcine deltacoronavirus and Porcine transmissible gastroenteritis virus
Abstract：Porcine deltacoronavirus (PDCoV) is a newly discovered coronavirus. It mainly infects piglets and causes severe enteritis accompanied by diarrhea and vomiting and other symptoms. The symptoms are symptomatically indistinguishable from those that caused by Porcine transmissible gastroenteritis virus (TGEV). In clinic the co-infection of PDCoV and TGEV is common, which causes huge economic losses to the swine industry. In order to control and prevent effectively these diseases, development of rapid detection methods of PDCoV and TGEV is the primary task. Thus, according to the analysis of PDCoV and TGEV N gene sequences in GenBank, two pairs of primers targeting the N gene of PDCoV and TGEV were designed respectively. By optimizing the PCR reaction conditions, a duplex RT-PCR detection method was established for the simultaneous detection of PDCoV and TGEV, and the specificity, sensitivity and repeatability of the method were studied. The results showed that the duplex RT-PCR had high sensitivity and specificity with a limited detection of 3.14×102 copies/μL for PDCoV and 3.68×103 copies/μL for TGEV, respectively, and no specific amplifications from other virus, such as Porcine epidemic diarrhea virus (PEDV), Porcine bocavirus (PBoV), Porcine respiratory and reproductive syndrome virus (PRRSV) or Porcine pseudorabies virus (PRV). 252 clinical samples tested by the duplex RT-PCR showed that the positive rates of PDCoV and TGEV were 12.30% and 4.76%, respectively, co-infection rate of PDCoV and TGEV was 0.79%. The date showed that the duplex RT-PCR method established in this study could be used for the clinical detection of PDCoV and TGEV, which provides the theoretical basis information and technical support for further applied to clinical differential diagnosis and epidemiological investigation of TGEV and PDCoV.
李小康, 赵丽, 崔保安,等. 2007. 多重PCR检测猪细小病毒和猪伪狂犬病病毒的研究[J]. 中国预防兽医学报, 29(03): 227-230.(Li X K, Zhao L, Cui B A, et al. 2007. Detection of porcine parvovirus and porcine pseudorabies virus by multiplex polymerase chain reaction. [J]. Chinese Journal of Preventive Veterinary Medicine, 29(03): 227-230.)Douglas M, Lindsey R, Yin J, et al. 2014, Rapid detection, complete genome sequencing, and phylogenetic analysis of porcine deltacoronavirus.[J]. Emerging Infectious Diseases, 20(8):1347-1350.Homwong N, Jarvis M C, Lam H C, et al. 2015. Characterization and evolution of porcine deltacoronavirus in the United States[J]. Preventive Veterinary Medicine, 23(2):435-444.Hu H, Jung K, Vlasova AN, et al. 2015, Isolation and Characterization of Porcine Deltacoronavirus from Pigs with Diarrhea in the United States [J]. J Clin Microbiol, 21(4): 1537-154.Jung K, Hu H, Eyerly B, et al. 2015. Pathogenicity of 2 Porcine Deltacoronavirus Strains in Gnotobiotic Pigs [J]. Emerging Infectious Diseases, 21(4): 650-654. Lee S, Lee C. 2014. Complete genome characterization of Korean porcine deltacoronavirus strain KOR/KNU14-04/2014. Genome Announc 2(6): e01191-14.Li G, Chen Q, Harmon K M, et al. 2014, Full-length genome sequence of porcine deltacoronavirus strain USA/IA/2014/8734[J]. Genome announcements, 2(2): e00278-14.Marthaler D, Raymond L, Jiang Y, et al. 2014. Rapid detection, complete genome sequencing, and phylogenetic analysis of porcine deltacoronavirus.[J]. Emerging Infectious Diseases, 20(8):1347-50.Ma Y, Zhang Y, Liang X, et al. 2015, Origin, evolution, and virulence of porcine deltacoronaviruses in the United States[J]. MBio, 6(2): e00064-15.Song D S, Kang B K, Oh J S, et al. 2006. Multiplex reverse transcription-PCR for rapid differential detection of porcine epidemic diarrhea virus, transmissible gastroenteritis virus, and porcine group A rotavirus.[J]. Journal of Veterinary Diagnostic Investigation Official Publication of the American Association of Veterinary Laboratory Diagnosticians Inc, 18(3):278-81.Wang L, Byrum B, Zhang Y. 2014, Detection and genetic characterization of deltacoronavirus in pigs, Ohio, USA, 2014.[J]. Emerging Infectious Diseases, 20(7):1227-30.Woo PC, Lau SK, Lam CS, et al. 2012. Discovery ofseven novel mammalian and avian coronaviruses in the genus Deltacoronavirussupports bat coronaviruses as the gene source of Alphacoronavirus and Betacoronavirus and avian coronaviruses as the genesource of Gammacoronavirus and Deltacoronavirus. [J]. Journal of Virology. 86:3995- 4008.Zhao J, Shi B J, Huang X G, et al. 2013. A multiplex RT-PCR assay for rapid and differential diagnosis of four porcine diarrhea associated viruses in field samples from pig farms in East China from 2010 to 2012 [J]. Journal of Virological Methods, 194(1–2):107-112.
