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


Pyöriä, Lari; Jokinen, Maija; Toppinen, Mari; Salminen, Henri; Vuorinen, Tytti; Hukkanen, Veijo; Schmotz, Constanze; Elbasani, Endrit; Ojala, Päivi M; Hedman, Klaus; Välimaa, Hannamari; Perdomo, Maria F (2020). HERQ-9 Is a New Multiplex PCR for Differentiation and Quantification of All Nine Human Herpesviruses. mSphere, 5(3):e00265-20.

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.

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.

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Item Type:Journal Article, refereed, original work
Communities & Collections:07 Faculty of Science > Institute of Evolutionary Biology and Environmental Studies
Dewey Decimal Classification:570 Life sciences; biology
590 Animals (Zoology)
Scopus Subject Areas:Life Sciences > Microbiology
Life Sciences > Molecular Biology
Language:English
Date:24 June 2020
Deposited On:16 Feb 2021 13:29
Last Modified:17 Feb 2021 21:01
Publisher:American Society for Microbiology
ISSN:2379-5042
OA Status:Gold
Free access at:Publisher DOI. An embargo period may apply.
Publisher DOI:https://doi.org/10.1128/msphere.00265-20

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