Donoso Mantke Oliver, Kallies René, Niklasson Bo, Nitsche Andreas, Niedrig Matthias
Centre for Biological Safety (ZBS-1), Robert Koch-Institut, Nordufer 20, D-13353 Berlin, Germany.
J Virol Methods. 2007 Apr;141(1):71-7. doi: 10.1016/j.jviromet.2006.11.029. Epub 2006 Dec 28.
Ljungan virus (LV), a new member of the Picornaviridae, recently isolated from vole species in both Sweden and the USA, is suspected to be pathogenic for humans as an aetiological agent in myocarditis, diabetes, neurological disease and perinatal disease. This study describes for the first time an RT-PCR assay that can identify and quantify LV infection in various tissue sample types using primers and two different minor-groove-binder probes targeting the 5'-untranslated region of the LV genome. The assay, evaluated using control samples derived from various virus cultures and rodent tissues, allows precise quantification of viral load over six orders of magnitude (10(1) to 10(6) viral copies per assay) for all known strains of LV with high sensitivity and specificity. Furthermore, a melting curve analysis (MCA) was developed using two amplicon-specific hybridisation probes that allows rapid genotyping of different LV strains. These new methods provide useful tools to investigate the putative role of LV as a pathogen in both rodents and humans.
吕永病毒（LV）是小核糖核酸病毒科的一个新成员，最近在瑞典和美国均从田鼠物种中分离得到，被怀疑作为心肌炎、糖尿病、神经疾病和围产期疾病的病原体对人类致病。本研究首次描述了一种逆转录聚合酶链反应（RT-PCR）检测方法，该方法可使用针对LV基因组5'非翻译区的引物和两种不同的小沟结合探针，对各种组织样本类型中的LV感染进行鉴定和定量。使用源自各种病毒培养物和啮齿动物组织的对照样本对该检测方法进行评估，对于所有已知的LV毒株，该方法能够以高灵敏度和特异性在六个数量级（每次检测10¹至10⁶个病毒拷贝）范围内精确量化病毒载量。此外，利用两种扩增子特异性杂交探针开发了熔解曲线分析（MCA）方法，可对不同的LV毒株进行快速基因分型。这些新方法为研究LV作为啮齿动物和人类病原体的假定作用提供了有用的工具。
