Diagnostic Laboratory, School of Veterinary Medicine, Faculty of Health Sciences, Aristotle University of Thessaloniki, 11 Stavrou Voutyra Str., 54627, Thessaloniki, Greece.
Laboratory of Pathology, School of Veterinary Medicine, Faculty of Health Sciences, Aristotle University of Thessaloniki, University Campus, 54124, Thessaloniki, Greece.
Mol Cell Probes. 2020 Jun;51:101528. doi: 10.1016/j.mcp.2020.101528. Epub 2020 Jan 28.
Small ruminant lentiviruses (SRLVs) are highly diverse retroviruses infecting sheep and goats. Although PCR-based testing is being utilized for diagnostics, its application is hampered by various factors. These include, among others, the exceptionally high genetic variability of SRLVs, as well as the low number of infected blood monocytes. For this reason, a highly sensitive and specific semi-nested real-time PCR for proviral DNA detection and quantification was developed. The method is innovative in that a) its design is based on selecting the preferred codon usage in the targeted conserved genomic regions and b) oligospermine-conjugated degenerate primers with increased Tm were utilized. Modifications permitted primer/template duplex formation in the cases of mismatches due to sporadic nucleotide polymorphisms in a number of variant SRLV strains and consequently, the detection of highly diverse SRLV strains. The potential loss of analytical sensitivity and specificity was counterbalanced by including a semi-nested step in combination with LNA probes. An in silico procedure for the evaluation of hybridization efficiency of the designed oligonucleotides to all known targeted variants was also implemented. The method presents a linear range of quantification over a 3-log range and a limit of detection of 3.9 proviral dsDNA copies per reaction. Its diagnostic performance was evaluated by testing field samples from seropositive and seronegative animals, followed by phylogenetic analysis of the strains detected. To further increase the diagnostic sensitivity, a DNA extraction protocol for blood leukocytes was developed and evaluated. A minimum of 500 ng input DNA is recommended for PCR-based detection of SRLV proviral DNA, given the low numbers of infected blood monocytes. The developed methodology may serve as a useful tool, which can be adjusted for the quantitative detection of viruses exhibiting high genetic variability.
小反刍兽瘟病毒（SRLV）是高度多样化的感染绵羊和山羊的逆转录病毒。虽然基于 PCR 的检测方法已被用于诊断，但由于各种因素的影响，其应用受到了限制。这些因素包括 SRLV 极高的遗传变异性，以及感染的血液单核细胞数量较少。因此，开发了一种高度敏感和特异的半巢式实时 PCR 用于检测和定量前病毒 DNA。该方法的创新性在于：a）其设计基于选择目标保守基因组区域中首选密码子使用；b）使用带有增加 Tm 的寡聚精胺缀合的简并引物。通过对多个变异 SRLV 株中由于散在的核苷酸多态性导致的引物/模板双链形成进行修饰，该方法能够检测到高度多样化的 SRLV 株。通过包括半巢式步骤和 LNA 探针来平衡分析灵敏度和特异性的潜在损失。还实施了一种用于评估设计的寡核苷酸与所有已知靶向变体杂交效率的计算机程序。该方法在 3 个对数范围内呈现定量线性范围，检测限为每个反应 3.9 个前病毒 dsDNA 拷贝。通过对来自血清阳性和血清阴性动物的田间样本进行测试，并对检测到的菌株进行系统发育分析，评估了该方法的诊断性能。为了进一步提高诊断灵敏度，开发并评估了一种用于血液白细胞的 DNA 提取方案。鉴于感染的血液单核细胞数量较少，建议用于 SRLV 前病毒 DNA 的 PCR 检测的最小输入 DNA 量为 500ng。开发的方法学可以作为一种有用的工具，可用于调整以用于检测具有高度遗传变异性的病毒的定量检测。
