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-log10 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.

中文翻译：

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利用寡精胺偶联的简并引物进行高灵敏的半巢式实时PCR，用于检测小型反刍动物慢病毒的各种菌株。

小型反刍动物慢病毒（SRLV）是感染绵羊和山羊的高度多样的逆转录病毒。尽管基于PCR的测试已用于诊断，但其应用受到各种因素的阻碍。这些尤其包括SRLV的遗传变异性极高，以及感染的血液单核细胞数量少。因此，开发了用于原病毒DNA检测和定量的高度灵敏，特异的半巢式实时PCR。该方法的创新之处在于：a）其设计基于在目标保守基因组区域中选择优选的密码子使用，以及b）使用Tm增加的寡精胺偶联的简并引物。在由于多种变异SRLV株中的零星核苷酸多态性而导致错配的情况下，修饰允许引物/模板双链体的形成，因此可以检测高度多样化的SRLV株。通过将半嵌套步骤与LNA探针结合使用，可以抵消分析灵敏度和特异性的潜在损失。还实施了计算机程序，用于评估设计的寡核苷酸与所有已知靶向变体的杂交效率。该方法显示了3-log10范围内的线性定量范围，并且每个反应的检出限为3.9个原病毒dsDNA拷贝。通过测试血清反应阳性和血清阴性动物的野外样品，然后对检测到的菌株进行系统发育分析，评估其诊断性能。为了进一步提高诊断灵敏度，开发并评估了用于血液白细胞的DNA提取方案。考虑到受感染的血液单核细胞数量少，建议至少500 ng输入DNA用于基于PCR的SRLV前病毒DNA检测。所开发的方法学可以用作有用的工具，可以对其进行调整以用于定量检测表现出高遗传变异性的病毒。