Quadruplex real-time PCR for rapid detection of human alphaherpesviruses
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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.
KeywordsHuman herpesvirus DNA Simultaneous detection
The authors would like to thank Veronika Fröhlich and Frieda Schön (both Kiel) as well as Heike Urban (Jena) for their excellent technical assistance, and Jennifer Bunke (Bonn) for critical reading of the manuscript. The kind support given by Professor Dr. Heinz Zeichhardt and Dr. Hans-Peter Grunert (both Instand e.V.) is kindly acknowledged. Furthermore, authors are indebted to Dr. Barbara Pohl (biomers.net), Mirjam Schwansee (Qiagen) and Jürgen Boelter (Roche) for stimulating discussion and advice.
No financial support or funding was received for this study.
Compliance with ethical standards
Conflict of interest
Biomers, Qiagen and Roche had no influence on interpretation of data and writing of the manuscript. A.K. was an invited speaker at the “Roche Tage 2018”. The authors act independently of these companies and declare no further conflict of interest.
This study exclusively included residual patient specimen initially sent for alphaherpesvirus diagnostics. Samples were only used anonymized. For this type of study formal consent is not required. The setting was approved by the Ethics Committee of the Christian-Albrechts-Universität zu Kiel.
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