Quadruplex real-time PCR for rapid detection of human alphaherpesviruses
- 26 Downloads
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.
- 1.Efstathiou S, Minson AC, Field HJ, Anderson JR, Wildy P (1986) Detection of herpes simplex virus-specific DNA sequences in latently infected mice and in humans. J Virol 57(2):446–455Google Scholar
- 3.Sauerbrei A, Schmitt S, Scheper T, Brandstadt A, Saschenbrecker S, Motz M, Soutschek E, Wutzler P (2011) Seroprevalence of herpes simplex virus type 1 and type 2 in Thuringia, Germany, 1999 to 2006. Euro Surveill 16:(44)Google Scholar
- 4.Wutzler P, Farber I, Wagenpfeil S, Bisanz H, Tischer A (2001) Seroprevalence of varicella-zoster virus in the German population. Vaccine 20(1–2):121–124Google Scholar
- 9.Pillet S, Verhoeven PO, Epercieux A, Bourlet T, Pozzetto B (2015) Development and validation of a laboratory-developed multiplex real-time PCR assay on the BD max system for detection of herpes simplex virus and varicella-zoster virus DNA in various clinical specimens. J Clin Microbiol 53(6):1921–1926. https://doi.org/10.1128/JCM.03692-14 Google Scholar
- 10.Wong AA, Pabbaraju K, Wong S, Tellier R (2016) Development of a multiplex real-time PCR for the simultaneous detection of herpes simplex and varicella zoster viruses in cerebrospinal fluid and lesion swab specimens. J Virol Methods 229:16–23. https://doi.org/10.1016/j.jviromet.2015.12.009 Google Scholar
- 12.Sauerbrei A, Eichhorn U, Hottenrott G, Wutzler P (2000) Virological diagnosis of herpes simplex encephalitis. J Clin Virol 17(1):31–36Google Scholar
- 13.Sauerbrei A, Eichhorn U, Schacke M, Wutzler P (1999) Laboratory diagnosis of herpes zoster. J Clin Virol 14(1):31–36Google Scholar
- 14.Cardenas AM, Edelstein PH, Alby K (2014) Development and optimization of a real-time PCR assay for detection of herpes simplex and varicella-zoster viruses in skin and mucosal lesions by use of the BD Max open system. J Clin Microbiol 52(12):4375–4376. https://doi.org/10.1128/JCM.02237-14 Google Scholar
- 15.Payan C, Ducancelle A, Aboubaker MH, Caer J, Tapia M, Chauvin A, Peyronnet D, Le Hen E, Arab Z, Legrand MC, Tran A, Postec E, Tourmen F, Avenel M, Malbois C, De Brux MA, Descamps P, Lunel F (2007) Human papillomavirus quantification in urine and cervical samples by using the Mx4000 and light cycler general real-time PCR systems. J Clin Microbiol 45(3):897–901. https://doi.org/10.1128/JCM.02022-06 Google Scholar
- 16.Filen F, Strand A, Allard A, Blomberg J, Herrmann B (2004) Duplex real-time polymerase chain reaction assay for detection and quantification of herpes simplex virus type 1 and herpes simplex virus type 2 in genital and cutaneous lesions. Sex Transm Dis 31(6):331–336Google Scholar
- 17.Pevenstein SR, Williams RK, McChesney D, Mont EK, Smialek JE, Straus SE (1999) Quantitation of latent varicella-zoster virus and herpes simplex virus genomes in human trigeminal ganglia. J Virol 73(12):10514–10518Google Scholar
- 18.Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, Mueller R, Nolan T, Pfaffl MW, Shipley GL, Vandesompele J, Wittwer CT (2009) The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem 55(4):611–622. https://doi.org/10.1373/clinchem.2008.112797 Google Scholar
- 20.Sauerbrei A, Wutzler P (2002) Laboratory diagnosis of central nervous system infections caused by herpesviruses. J Clin Virol 25(Suppl 1):S45–S51Google Scholar
- 22.Espy MJ, Ross TK, Teo R, Svien KA, Wold AD, Uhl JR, Smith TF (2000) Evaluation of LightCycler PCR for implementation of laboratory diagnosis of herpes simplex virus infections. J Clin Microbiol 38(8):3116–3118Google Scholar
- 23.Espy MJ, Teo R, Ross TK, Svien KA, Wold AD, Uhl JR, Smith TF (2000) Diagnosis of varicella-zoster virus infections in the clinical laboratory by LightCycler PCR. J Clin Microbiol 38(9):3187–3189Google Scholar
- 24.Espy MJ, Uhl JR, Mitchell PS, Thorvilson JN, Svien KA, Wold AD, Smith TF (2000) Diagnosis of herpes simplex virus infections in the clinical laboratory by LightCycler PCR. J Clin Microbiol 38(2):795–799Google Scholar
- 25.Stocher M, Leb V, Bozic M, Kessler HH, Halwachs-Baumann G, Landt O, Stekel H, Berg J (2003) Parallel detection of five human herpes virus DNAs by a set of real-time polymerase chain reactions in a single run. J Clin Virol 26(1):85–93Google Scholar
- 26.Burrows J, Nitsche A, Bayly B, Walker E, Higgins G, Kok T (2002) Detection and subtyping of Herpes simplex virus in clinical samples by LightCycler PCR, enzyme immunoassay and cell culture. BMC Microbiol 2:12Google Scholar
- 32.Chaudhuri A, Kennedy PG (2002) Diagnosis and treatment of viral encephalitis. Postgrad Med J 78(924):575–583Google Scholar
- 34.Frisk AL, Konig M, Moritz A, Baumgartner W (1999) Detection of canine distemper virus nucleoprotein RNA by reverse transcription-PCR using serum, whole blood, and cerebrospinal fluid from dogs with distemper. J Clin Microbiol 37(11):3634–3643Google Scholar
- 35.Ninove L, Nougairede A, Gazin C, Thirion L, Delogu I, Zandotti C, Charrel RN, De Lamballerie X (2011) RNA and DNA bacteriophages as molecular diagnosis controls in clinical virology: a comprehensive study of more than 45,000 routine PCR tests. PLoS One 6(2):e16142. https://doi.org/10.1371/journal.pone.0016142 Google Scholar