Molecular analysis of virus genomes/transcript in the intraocular fluid samples during viral uveitis

在这项研究中，我们探讨了定量诊断方法用于快速诊断和治疗病毒性眼病。特别是巨细胞病毒（CMV）视网膜炎和急性视网膜坏死。应用真实的实时聚合酶链反应（PCR）技术，检测了人巨细胞病毒（CMV）和水痘-带状疱疹病毒（VZV）的基因组含量。我们发现病毒基因组的拷贝数与病变活性之间呈正相关。这些拷贝数在抗病毒治疗后迅速减少。1例CMV视网膜炎后免疫恢复性葡萄膜炎患者玻璃体液中CMV基因组拷贝数低于检测水平，但可检测到大量病毒抗原。下一步，我们将利用新近发展的环介导等温扩增（LAMP）技术进一步研究一种更快速、特异、简便的诊断方法。使用这种LAMP方法，我们确定了急性视网膜坏死患者中VZV的病毒基因组，并发现进行这种分析是可靠的。该方法与实时荧光定量PCR检测结果相关性较好，但灵敏度低于真实的实时荧光定量PCR。除一份标本外，其他标本均可以直接进行LAMP分析。因此，LAMP方法具有半定量、简便、快速的特点。因此，该方法对病毒性眼病的临床诊断具有一定的应用价值。

In this study we investigated the quantitative diagnostic methods useful for rapid diagnosis and treatment of viral diseases of the eye. Especially, we focused on the cytomegalovirus (CMV) retinitis and acute retinal necrosis. Using the real time polymerase chain reaction (PCR) we determined the amount of the viral genomes of human CMV and varicella-zoster virus (VZV) in fluid samples aspirated from the eyeballs. We found the positive correlation between the copy numbers of the viral genomes and the lesion activity. These copy numbers became rapidly decreased after the antiviral treatment. In one patient with immune recovery uveitis after CMV retinitis, the copy number of human CMV genome in the vitreous fluid was less than the level of detection, but the high amount of viral antigens could he detected. As next step we further studies a more prompt, specific and simple diagnostic method using a recently developed loop-mediated isothermal amplification (LAMP). Using this LAMP method we determined the viral genomes of VZV in patients with acute retinal necrosis and found to be reliable to do this analysis. The result by this method was well correlated with that by real-time PCR, but the sensitively was less than that by real time PCR. In addition, we could apply the samples without DNA extraction for LAMP analysis except one specimen. In conclusion, LAMP method is semi-quantitative but simple and rapid. From this conclusion, this method will he valuable for the clinical application for diagnosis of viral ocular diseases.