Infections with the nine human herpesviruses (HHVs) are globally prevalent and characterized by lifelong persistence. Reactivations can potentially manifest as life-threatening conditions for which the demonstration of viral DNA is essential. In the present study, we developed HERQ-9, a pan-HHV quantitative PCR designed in triplex reactions to differentiate and quantify each of the HHV-DNAs: (i) herpes simplex viruses 1 and 2 and varicella-zoster virus; (ii) Epstein-Barr virus, human cytomegalovirus, and Kaposi’s sarcoma-associated herpesvirus; and (iii) HHV-6A, -6B, and -7. The method was validated with prequantified reference standards as well as with mucocutaneous swabs and cerebrospinal fluid, plasma, and tonsillar tissue samples. Our findings highlight the value of multiplexing in the diagnosis of many unsuspected, yet clinically relevant, herpesviruses. In addition, we report here frequent HHV-DNA co-occurrences in clinical samples, including some previously unknown. HERQ-9 exhibited high specificity and sensitivity (LOD₉₅s of ∼10 to ∼17 copies/reaction), with a dynamic range of 10¹ to 10⁶ copies/μl. Moreover, it performed accurately in the coamplification of both high- and low-abundance targets in the same reaction. In conclusion, we demonstrated that HERQ-9 is suitable for the diagnosis of a plethora of herpesvirus-related diseases. Besides its significance to clinical management, the method is valuable for the assessment of hitherto-unexplored synergistic effects of herpesvirus coinfections. Furthermore, its high sensitivity enables studies on the human virome, often dealing with minute quantities of persisting HHVs.

中文翻译：

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HERQ-9是一种用于鉴别和定量所有九种人类疱疹病毒的新型多重PCR。

九种人类疱疹病毒（HHV）的感染在全球范围内普遍存在，并且具有终身持久性。重新激活可能表现为威胁生命的条件，对此病毒DNA的展示至关重要。在本研究中，我们开发了HERQ-9，这是一种通过三重反应设计的泛HHV定量PCR，用于区分和定量每种HHV-DNA：（i）单纯疱疹病毒1和2和水痘带状疱疹病毒；（ii）爱泼斯坦-巴尔病毒，人巨细胞病毒和卡波济氏肉瘤相关疱疹病毒；（iii）HHV-6A，-6B和-7。该方法已通过预先量化的参考标准以及粘膜皮肤拭子和脑脊液，血浆和扁桃体组织样品进行了验证。我们的发现强调了多重诊断在许多未曾怀疑但仍具有临床意义的诊断中的价值，疱疹病毒。此外，我们在这里报告临床样品中频繁发生HHV-DNA共存，包括一些以前未知的。HERQ-9表现出高特异性和敏感性（LOD_95个S的〜10至~17拷贝/反应），用10的动态范围¹至10 ^6个拷贝/μl的。而且，它可以在同一反应中高丰度和低丰度目标的共扩增中准确执行。总之，我们证明了HERQ-9适用于多种疱疹病毒相关疾病的诊断。除了对临床管理的意义外，该方法对于评估疱疹病毒共感染迄今尚未探索的协同作用也很有价值。此外，它的高灵敏度使得能够对人类病毒进行研究，从而经常处理微量的持续性HHV。