HERQ-9 Is a New Multiplex PCR for Differentiation and Quantification of All Nine Human Herpesviruses

ABSTRACT 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...

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Autores principales:	Lari Pyöriä, Maija Jokinen, Mari Toppinen, Henri Salminen, Tytti Vuorinen, Veijo Hukkanen, Constanze Schmotz, Endrit Elbasani, Päivi M. Ojala, Klaus Hedman, Hannamari Välimaa, Maria F. Perdomo
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2020
Materias:	Epstein-Barr virus HHV-6 coinfection cytomegalovirus diagnostics human herpesviruses Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/a6c28107b8b94c3c8fc20a1839e4bf27
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spelling	oai:doaj.org-article:a6c28107b8b94c3c8fc20a1839e4bf272021-11-15T15:30:16ZHERQ-9 Is a New Multiplex PCR for Differentiation and Quantification of All Nine Human Herpesviruses10.1128/mSphere.00265-202379-5042https://doaj.org/article/a6c28107b8b94c3c8fc20a1839e4bf272020-06-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mSphere.00265-20https://doaj.org/toc/2379-5042ABSTRACT 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. IMPORTANCE By adulthood, almost all humans become infected by at least one herpesvirus (HHV). The maladies inflicted by these microbes extend beyond the initial infection, as they remain inside our cells for life and can reactivate, causing severe diseases. The diagnosis of active infection by these ubiquitous pathogens includes the detection of DNA with sensitive and specific assays. We developed the first quantitative PCR assay (HERQ-9) designed to identify and quantify each of the nine human herpesviruses. The simultaneous detection of HHVs in the same sample is important since they may act together to induce life-threatening conditions. Moreover, the high sensitivity of our method is of extreme value for assessment of the effects of these viruses persisting in our body and their long-term consequences on our health.Lari PyöriäMaija JokinenMari ToppinenHenri SalminenTytti VuorinenVeijo HukkanenConstanze SchmotzEndrit ElbasaniPäivi M. OjalaKlaus HedmanHannamari VälimaaMaria F. PerdomoAmerican Society for MicrobiologyarticleEpstein-Barr virusHHV-6coinfectioncytomegalovirusdiagnosticshuman herpesvirusesMicrobiologyQR1-502ENmSphere, Vol 5, Iss 3 (2020)
institution	DOAJ
collection	DOAJ
language	EN
topic	Epstein-Barr virus HHV-6 coinfection cytomegalovirus diagnostics human herpesviruses Microbiology QR1-502
spellingShingle	Epstein-Barr virus HHV-6 coinfection cytomegalovirus diagnostics human herpesviruses Microbiology QR1-502 Lari Pyöriä Maija Jokinen Mari Toppinen Henri Salminen Tytti Vuorinen Veijo Hukkanen Constanze Schmotz Endrit Elbasani Päivi M. Ojala Klaus Hedman Hannamari Välimaa Maria F. Perdomo HERQ-9 Is a New Multiplex PCR for Differentiation and Quantification of All Nine Human Herpesviruses
description	ABSTRACT 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. IMPORTANCE By adulthood, almost all humans become infected by at least one herpesvirus (HHV). The maladies inflicted by these microbes extend beyond the initial infection, as they remain inside our cells for life and can reactivate, causing severe diseases. The diagnosis of active infection by these ubiquitous pathogens includes the detection of DNA with sensitive and specific assays. We developed the first quantitative PCR assay (HERQ-9) designed to identify and quantify each of the nine human herpesviruses. The simultaneous detection of HHVs in the same sample is important since they may act together to induce life-threatening conditions. Moreover, the high sensitivity of our method is of extreme value for assessment of the effects of these viruses persisting in our body and their long-term consequences on our health.
format	article
author	Lari Pyöriä Maija Jokinen Mari Toppinen Henri Salminen Tytti Vuorinen Veijo Hukkanen Constanze Schmotz Endrit Elbasani Päivi M. Ojala Klaus Hedman Hannamari Välimaa Maria F. Perdomo
author_facet	Lari Pyöriä Maija Jokinen Mari Toppinen Henri Salminen Tytti Vuorinen Veijo Hukkanen Constanze Schmotz Endrit Elbasani Päivi M. Ojala Klaus Hedman Hannamari Välimaa Maria F. Perdomo
author_sort	Lari Pyöriä
title	HERQ-9 Is a New Multiplex PCR for Differentiation and Quantification of All Nine Human Herpesviruses
title_short	HERQ-9 Is a New Multiplex PCR for Differentiation and Quantification of All Nine Human Herpesviruses
title_full	HERQ-9 Is a New Multiplex PCR for Differentiation and Quantification of All Nine Human Herpesviruses
title_fullStr	HERQ-9 Is a New Multiplex PCR for Differentiation and Quantification of All Nine Human Herpesviruses
title_full_unstemmed	HERQ-9 Is a New Multiplex PCR for Differentiation and Quantification of All Nine Human Herpesviruses
title_sort	herq-9 is a new multiplex pcr for differentiation and quantification of all nine human herpesviruses
publisher	American Society for Microbiology
publishDate	2020
url	https://doaj.org/article/a6c28107b8b94c3c8fc20a1839e4bf27
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HERQ-9 Is a New Multiplex PCR for Differentiation and Quantification of All Nine Human Herpesviruses

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