Labor Dr. Krause und Kollegen MVZ GmbH Kiel, Steenbeker Weg 23, 24106, Kiel, Germany.
Institut für Infektionsmedizin, Christian-Albrechts-Universität zu Kiel und Universitätsklinikum Schleswig-Holstein, Campus Kiel, Brunswiker Strasse 4, 24105, Kiel, Germany.
Med Microbiol Immunol. 2019 Apr;208(2):197-204. doi: 10.1007/s00430-019-00580-2. Epub 2019 Jan 24.
Infections with the herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) as well as with the varicella-zoster virus (VZV) may take a serious course. Thus, rapid and reliable detection of these alphaherpesviruses is urgently needed. For this, we established a qualitative quadruplex real-time polymerase chain reaction (PCR) covering HSV-1, HSV-2, VZV and endogenous human glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The PCR was validated with quality assessment samples and pre-characterized clinical samples including swabs, blood and cerebrospinal as well as respiratory fluids. For comparison, nucleic acids (NA) of selected samples were extracted manually and automatically. The protocol takes approx. 90 min, starting with the preparation of NA until the report of results. The oligonucleotide and hydrolysis probe sequences specifically detect and distinguish HSV-1 (530 nm), HSV-2 (705 nm) and VZV (560 nm) DNA. The detection limit was estimated with 100-500 copies/ml HSV-1 and HSV-2/VZV, respectively. All quality assessment samples as well as all the patient samples were classified correctly. Parallel detection of GAPDH (670 nm) DNA was implemented to demonstrate correct sampling, but was uncertain in case of swabs. To this end, alphaherpesvirus-free human DNA was also added directly into the mastermix to exclude PCR inhibition. The established protocol for parallel detection and differentiation of alphaherpesviruses is fast, highly specific as well as rather sensitive. It will facilitate HSV-1/2 and VZV diagnostics and may be further improved by opening the 670 nm channel for a combined extraction and PCR inhibition control.
单纯疱疹病毒 1 型（HSV-1）和 2 型（HSV-2）以及水痘-带状疱疹病毒（VZV）感染可能会导致严重后果。因此，迫切需要快速、可靠地检测这些α疱疹病毒。为此，我们建立了一种定性四重实时聚合酶链反应（PCR），涵盖 HSV-1、HSV-2、VZV 和内源性人甘油醛 3-磷酸脱氢酶（GAPDH）。该 PCR 使用质量评估样本和预特征化的临床样本（包括拭子、血液和脑脊液以及呼吸道液）进行了验证。为了比较，手动和自动提取了选定样本的核酸（NA）。该方案从 NA 的制备开始，到报告结果，大约需要 90 分钟。寡核苷酸和水解探针序列特异性检测和区分 HSV-1（530nm）、HSV-2（705nm）和 VZV（560nm）DNA。HSV-1 和 HSV-2/VZV 的检测限分别估计为 100-500 拷贝/ml。所有质量评估样本和所有患者样本均被正确分类。同时检测 GAPDH（670nm）DNA 以证明正确采样，但在拭子情况下不确定。为此，还直接将无α疱疹病毒的人 DNA 加入主混合物中以排除 PCR 抑制。建立的用于平行检测和区分α疱疹病毒的方案快速、高度特异性且相当敏感。它将促进 HSV-1/2 和 VZV 诊断，并且通过打开 670nm 通道进行联合提取和 PCR 抑制控制，可能会进一步改进。
