■Capropoxvirus(CaPV)属的三个成员,包括块状皮肤病病毒(LSDV),山羊痘病毒(GTPV),羊痘病毒(SPPV),被世界动物卫生组织列为应呈报形式。这些病毒对全世界的反刍动物养殖业产生了负面影响,造成巨大的经济损失。尽管SPPV和GTPV仅在一种动物中引起更严重的临床疾病,它们可以在绵羊和山羊之间转移。同源和异源免疫策略均用于保护动物免受CaPV的侵害。然而,发展准确快速的方法来区分这三种病毒有助于早期发现,疾病监测,和控制CaPV感染。因此,我们开发了一种新的三重实时PCR(qPCR)用于LSDV的分化,GTPV,和SPPV。
■设计通用引物来检测泛CaPV序列。设计了基于物种特异性小沟粘合剂(MGB)的探针,用FAM标记LSDV,GTPV的十六进制,和ROX为SPPV。敏感性,特异性,再现性,并对混合感染的检测能力进行了三重qPCR评估。Further,226个感染和阴性对照的临床样品进行三重qPCR,并使用PCR-限制性片段长度多态性(PCR-RFLP)和PRO30基因的测序方法对结果进行了验证。
■三重qPCR可以成功区分LSDV,GTPV,和SPPV在一个反应中,检测灵敏度分别为5.41、27.70和17.28拷贝/μL,分别。没有观察到与其他引起常见反刍动物疾病的病毒的交叉反应,包括小反刍动物病毒,口蹄疫病毒,蓝舌病毒,绵羊传染性脓疱性皮炎病毒,传染性牛鼻支气管炎病毒,和牛病毒性腹泻粘膜病病毒。测定间和测定内变异性<2.5%。结果表明,三重qPCR具有高度特异性,敏感,和可重复的。模拟实验表明,在混合感染的情况下,该测定法可以成功区分两种或三种病毒,而没有任何交叉反应。对于临床样本,结果与PCR-RFLP和测序结果完全一致。这证明该测定对于临床应用是可靠的。
■三重qPCR是一种强大的,快速,和识别各种类型的CaPV的简单工具,因为它可以成功区分LSDV,GTPV,和SPPV在一个反应中。此外,该检测方法可以促进更准确的疾病诊断和监测,从而更好地控制CaPV感染。
UNASSIGNED: Three members of
Capripoxvirus (CaPV) genus, including lumpy skin disease virus (LSDV), goatpox virus (GTPV), and sheeppox virus (SPPV), are mentioned as notifiable forms by World Organization for Animal Health. These viruses have negatively impacted ruminant farming industry worldwide, causing great economic losses. Although SPPV and GTPV cause more severe clinical disease in only one animal species, they can transfer between sheep and goats. Both homologous and heterologous immunization strategies are used to protect animals against CaPVs. However, development of accurate and rapid methods to distinguish these three viruses is helpful for the early detection, disease surveillance, and control of CaPV infection. Therefore, we developed a novel triplex real-time PCR (qPCR) for the differentiation of LSDV, GTPV, and SPPV.
UNASSIGNED: Universal primers were designed to detect pan-CaPV sequences. Species-specific minor groove binder (MGB)-based probes were designed, which were labeled with FAM for LSDV, HEX for GTPV, and ROX for SPPV. The sensitivity, specificity, reproducibility, and ability of detecting mixed infections were evaluated for the triplex qPCR. Further, 226 clinical samples of the infection and negative controls were subjected to the triplex qPCR, and the results were verified using PCR-restriction fragment length polymorphism (PCR-RFLP) and sequencing methods for PRO30 gene.
UNASSIGNED: The triplex qPCR could successfully distinguish LSDV, GTPV, and SPPV in one reaction, and the assay sensitivity was 5.41, 27.70, and 17.28 copies/μL, respectively. No cross-reactivity was observed with other viruses causing common ruminant diseases, including des petits ruminants virus, foot-and-mouth disease virus, bluetongue virus, ovine contagious pustular dermatitis virus, infectious bovine rhinotracheitis virus, and bovine viral diarrhea-mucosal disease virus. Inter-and intra-assay variabilities were < 2.5%. The results indicated that the triplex qPCR was highly specific, sensitive, and reproducible. Simulation experiments revealed that this assay could successfully distinguish two or three viruses in case of mixed infections without any cross-reaction. For clinical samples, the results were completely consistent with the results of PCR-RFLP and sequencing. This demonstrated that the assay was reliable for clinical application.
UNASSIGNED: The triplex qPCR is a robust, rapid, and simple tool for identifying various types of CaPV as it can successfully distinguish LSDV, GTPV, and SPPV in one reaction. Furthermore, the assay can facilitate more accurate disease diagnosis and surveillance for better control of CaPV infection.