Gall A, Hoffmann B, Teifke J P, Lange B, Schirrmeier H
Friedrich-Loeffler-Institut, Institute of Diagnostic Virology, Boddenblick 5a, 17493 Greifswald-Insel Riems, Germany.
Vet Microbiol. 2007 Feb 25;120(1-2):17-32. doi: 10.1016/j.vetmic.2006.10.006. Epub 2006 Oct 12.
An internally controlled multiplex real-time RT-PCR using TaqMan probes and external standards for absolute RNA quantification was developed as a new diagnostic tool for the detection of rabbit haemorrhagic disease virus (RHDV). The test revealed a specificity of 100%, an analytical sensitivity of 10 copies/well and a linearity over a range from 10(1) to 10(10) copies. The viral loads in organs, leukocytes, sera and excretions of seropositive, convalescent rabbits which were overcoming an experimental infection with RHDV were determined using the validated assay. As a result, viral RNA was demonstrated and quantified for at least 15 weeks. Thus, a persistence of viral RNA after experimental infection of rabbits could be shown for the first time. In contrast, neither antigen nor infectious virus could be detected by antigen-ELISA, immunohistochemistry or experimental transmission. Therefore, further experiments are necessary to prove that the persistence of RNA is linked with the persistence of infectious virus particles.
开发了一种使用TaqMan探针和外标进行绝对RNA定量的内控多重实时RT-PCR技术，作为检测兔出血性疾病病毒（RHDV）的新型诊断工具。该检测方法显示特异性为100%，分析灵敏度为10拷贝/孔，在10¹至10¹⁰拷贝范围内呈线性。使用经过验证的检测方法测定了正在克服RHDV实验性感染的血清阳性、恢复期兔的器官、白细胞、血清和排泄物中的病毒载量。结果表明，病毒RNA至少在15周内得到了证实和定量。因此，首次证明了兔实验性感染后病毒RNA的持续存在。相比之下，通过抗原ELISA、免疫组织化学或实验性传播均未检测到抗原或传染性病毒。因此，需要进一步实验来证明RNA的持续存在与传染性病毒颗粒的持续存在有关。
