London Centre for Nanotechnology, Faculty of Maths and Physical Sciences, University College London, London, United Kingdom; Department of Clinical Virology, University College London Hospitals NHS Foundation Trust, London, United Kingdom.
Advanced Pathogen Diagnostics Unit, University College London Hospitals NHS Foundation Trust, London, United Kingdom.
J Virol Methods. 2019 Jun;268:17-23. doi: 10.1016/j.jviromet.2019.03.006. Epub 2019 Mar 11.
Dengue is a vector-transmitted viral infection that is a significant cause of morbidity and mortality in humans worldwide, with over 50 million apparent cases and a fatality rate of 2.5 % of 0.5 million severe cases per annum in recent years. Four serotypes are currently co-circulating. Diagnosis of infection may be by polymerase chain reaction, serology or rapid antigen test for NS1. Both pan-serotype and serotype-specific genome detection assays have been described, however, achieving adequate sensitivity with pan-serotype assays has been challenging. Indeed, as we show here, inspection of components and cycling parameters of a pan-serotype RT-qPCR assay in use in laboratories worldwide revealed insufficient probe stability to accommodate potential nucleotide mismatches, resulting in false-negatives. A minor-groove binder (MGB)-modified version of the probe was designed and its performance compared with that of the original probe in 32 samples. Eight of the samples were undetected by the original probe but detected by the MGB modified probe and six out of seven of these that could be serotyped belonged to serotype 4. Sequencing of the region targeted by the probe in these samples revealed two mismatches which were also universally present in all other serotype 4 sequences in a public database. We therefore recommend adoption of this MGB modification in order to reduce the risk of false-negative results, especially with dengue serotype 4 infections.
登革热是一种经媒介传播的病毒感染，是全球范围内导致发病率和死亡率的重要原因，近年来每年有超过 5000 万例显性病例和 0.5%的 50 万例重症病例死亡率。目前有 4 种血清型同时流行。感染的诊断可以通过聚合酶链反应、血清学或快速抗原检测 NS1 进行。已经描述了针对所有血清型和针对特定血清型的基因组检测方法，但是，实现针对所有血清型检测方法的足够灵敏度一直具有挑战性。事实上，正如我们在这里所展示的，对全球实验室使用的一种针对所有血清型 RT-qPCR 检测方法的组件和循环参数进行检查后发现，探针的稳定性不足以适应潜在的核苷酸错配，导致假阴性。设计了一种小沟结合物（MGB）修饰的探针，并将其与原始探针在 32 个样本中的性能进行了比较。原始探针未能检测到的 8 个样本被 MGB 修饰的探针检测到，其中 7 个可以进行血清型分类的样本中有 6 个属于血清型 4。对这些样本中探针靶向区域的测序揭示了两个错配，这些错配在公共数据库中所有其他血清型 4 序列中也普遍存在。因此，我们建议采用这种 MGB 修饰以降低假阴性结果的风险，尤其是在登革热血清型 4 感染的情况下。
