Woźniakowski Grzegorz, Niczyporuk Jowita Samanta
Department of Swine Diseases, National Veterinary Research Institute, Partyzantów 57 Avenue, 24-100 Puławy, Poland.
Department Poultry Viral Diseases, National Veterinary Research Institute, Partyzantów 57 Avenue, 24-100 Puławy, Poland.
J Virol Methods. 2015 Sep 1;221:22-8. doi: 10.1016/j.jviromet.2015.04.015. Epub 2015 Apr 25.
Marek's disease (MD) is a tumoral disease of chickens that can be controlled by vaccines based on non-pathogenic strains of turkey herpesvirus (HVT), SB-1 strain belonging to serotype 2, or the attenuated CVI988/Rispens strain belonging to serotype 1 of Marek's disease virus (MDV). Currently, the 'gold standard' in MD prophylaxis is the Rispens strain-based vaccine which protects against very virulent MDV and disease onset. Previous studies have shown that loop-mediated isothermal amplification (LAMP) is a rapid alternative to polymerase chain reaction (PCR) for detection and differentiation of HVT, SB-1 and virulent MDV strains. The aim of this study was to develop and evaluate a novel LAMP assay for the detection of the UL49 Rispens-specific region. This assay was validated using material from infected chicken embryo fibroblasts (CEFs) and tissue samples from vaccinated chickens. The analytical sensitivity of the assay was 10-times higher than PCR and reliably amplified 0.1 log10 TCID50/ml. The MDV Rispens was also detected at 18h after infection of CEFs. The results showed LAMP to be selective and a sensitive method to detect Rispens as early as 3 d.p.v. in all internal organs of chickens. Furthermore, the method was also capable to detect Rispens in 5 out of 26 chickens originating from different flocks. A mismatch amplification mutation assay (MAMA-PCR) confirmed the presence of Rispens strain in all LAMP-positive chickens. This is the first report of the specific visual detection of Rispens in vitro and in vivo using LAMP. The method may be useful for monitoring of successful chicken vaccination as well as in vitro studies in infected cell cultures.
马立克氏病（MD）是鸡的一种肿瘤性疾病，可通过基于火鸡疱疹病毒（HVT）非致病株的疫苗来控制，HVT的SB - 1株属于血清型2，或通过马立克氏病病毒（MDV）血清型1的减毒CVI988 / Rispens株来控制。目前，MD预防的“金标准”是基于Rispens株的疫苗，它可预防超强毒MDV和疾病的发生。先前的研究表明，环介导等温扩增技术（LAMP）是一种用于检测和区分HVT、SB - 1和强毒MDV株的快速替代聚合酶链反应（PCR）的方法。本研究的目的是开发和评估一种用于检测UL49 Rispens特异性区域的新型LAMP检测方法。该检测方法使用来自感染鸡胚成纤维细胞（CEF）的材料和接种疫苗鸡的组织样本进行了验证。该检测方法的分析灵敏度比PCR高10倍，能可靠地扩增0.1 log10 TCID50/ml。在CEF感染后18小时也检测到了MDV Rispens。结果表明，LAMP具有选择性，是一种在鸡的所有内脏器官中最早在接种后3天就能检测到Rispens的灵敏方法。此外，该方法还能够在来自不同鸡群的26只鸡中的5只中检测到Rispens。错配扩增突变分析（MAMA - PCR）证实了所有LAMP阳性鸡中都存在Rispens株。这是首次使用LAMP在体外和体内特异性可视化检测Rispens的报告。该方法可能有助于监测鸡疫苗接种的成功情况以及在感染细胞培养物中的体外研究。
