Cycling probe-based real-time PCR for the detection of Human herpesvirus 6A and B

Masaru Ihira, Ayumi Yamaki, Yuri Kato, Yuki Higashimoto, Yoshiki Kawamura, Tetsushi Yoshikawa

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

Human herpesvirus 6 (HHV-6) is classified as two distinct species: HHV-6A and B. HHV-6B infection can cause several clinical manifestations in transplant recipients including encephalitis, bone marrow suppression, and pneumonitis. In contrast to HHV-6B, the clinical features of HHV-6A infection remain largely undefined. Herein, we developed a multiplex cycling probe real-time PCR that discriminated between HHV-6A and HHV-6B. The assay was HHV-6-specific and no cross amplification was demonstrated for other herpesviruses. Moreover, the assay had a broad, linear dynamic range of detection between 1 and 106 copies of viral DNA. The quantification of HHV-6A DNA was suppressed by an excess amount of HHV-6B DNA (1 × 106 copies/tube) in the multiplex PCR assay; however, 1 × 106 copies/tube of HHV-6A DNA did not affect the quantification of 1 × 104 copies/tube of HHV-6B DNA. To determine the reliability of the assay for analysis of clinical specimens, DNAs extracted from the peripheral blood of hematopoietic stem cell transplant recipients were assayed using our multiplex real-time PCR versus the standard TaqMan PCR. Strong correlations were demonstrated between the two different assay systems for both HHV-6A (R2 = 0.913) and HHV-6B (R2 = 0.909). Therefore, our multiplex HHV-6 species-specific cycling probe real-time PCR is useful for evaluating the precise copy numbers of HHV-6A and B in transplant recipients. However, as HHV-6A copy numbers was affected by presence of high copies of HHV-6B DNA (1 × 106 copies/tube), it may be difficult to measure precise copy numbers of HHV-6A in inherited chromosomally integrated HHV-6B patient. J. Med. Virol. 88:1628–1635, 2016.

Original languageEnglish
Pages (from-to)1628-1635
Number of pages8
JournalJournal of Medical Virology
Volume88
Issue number9
DOIs
Publication statusPublished - 01-09-2016

Fingerprint

Human Herpesvirus 6
Cercopithecine Herpesvirus 1
Real-Time Polymerase Chain Reaction
DNA
Multiplex Polymerase Chain Reaction

All Science Journal Classification (ASJC) codes

  • Virology
  • Infectious Diseases

Cite this

Ihira, Masaru ; Yamaki, Ayumi ; Kato, Yuri ; Higashimoto, Yuki ; Kawamura, Yoshiki ; Yoshikawa, Tetsushi. / Cycling probe-based real-time PCR for the detection of Human herpesvirus 6A and B. In: Journal of Medical Virology. 2016 ; Vol. 88, No. 9. pp. 1628-1635.
@article{47ac37997e9c47e3b2bdfcaab7732e31,
title = "Cycling probe-based real-time PCR for the detection of Human herpesvirus 6A and B",
abstract = "Human herpesvirus 6 (HHV-6) is classified as two distinct species: HHV-6A and B. HHV-6B infection can cause several clinical manifestations in transplant recipients including encephalitis, bone marrow suppression, and pneumonitis. In contrast to HHV-6B, the clinical features of HHV-6A infection remain largely undefined. Herein, we developed a multiplex cycling probe real-time PCR that discriminated between HHV-6A and HHV-6B. The assay was HHV-6-specific and no cross amplification was demonstrated for other herpesviruses. Moreover, the assay had a broad, linear dynamic range of detection between 1 and 106 copies of viral DNA. The quantification of HHV-6A DNA was suppressed by an excess amount of HHV-6B DNA (1 × 106 copies/tube) in the multiplex PCR assay; however, 1 × 106 copies/tube of HHV-6A DNA did not affect the quantification of 1 × 104 copies/tube of HHV-6B DNA. To determine the reliability of the assay for analysis of clinical specimens, DNAs extracted from the peripheral blood of hematopoietic stem cell transplant recipients were assayed using our multiplex real-time PCR versus the standard TaqMan PCR. Strong correlations were demonstrated between the two different assay systems for both HHV-6A (R2 = 0.913) and HHV-6B (R2 = 0.909). Therefore, our multiplex HHV-6 species-specific cycling probe real-time PCR is useful for evaluating the precise copy numbers of HHV-6A and B in transplant recipients. However, as HHV-6A copy numbers was affected by presence of high copies of HHV-6B DNA (1 × 106 copies/tube), it may be difficult to measure precise copy numbers of HHV-6A in inherited chromosomally integrated HHV-6B patient. J. Med. Virol. 88:1628–1635, 2016.",
author = "Masaru Ihira and Ayumi Yamaki and Yuri Kato and Yuki Higashimoto and Yoshiki Kawamura and Tetsushi Yoshikawa",
year = "2016",
month = "9",
day = "1",
doi = "10.1002/jmv.24513",
language = "English",
volume = "88",
pages = "1628--1635",
journal = "Journal of Medical Virology",
issn = "0146-6615",
publisher = "Wiley-Liss Inc.",
number = "9",

}

Cycling probe-based real-time PCR for the detection of Human herpesvirus 6A and B. / Ihira, Masaru; Yamaki, Ayumi; Kato, Yuri; Higashimoto, Yuki; Kawamura, Yoshiki; Yoshikawa, Tetsushi.

In: Journal of Medical Virology, Vol. 88, No. 9, 01.09.2016, p. 1628-1635.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Cycling probe-based real-time PCR for the detection of Human herpesvirus 6A and B

AU - Ihira, Masaru

AU - Yamaki, Ayumi

AU - Kato, Yuri

AU - Higashimoto, Yuki

AU - Kawamura, Yoshiki

AU - Yoshikawa, Tetsushi

PY - 2016/9/1

Y1 - 2016/9/1

N2 - Human herpesvirus 6 (HHV-6) is classified as two distinct species: HHV-6A and B. HHV-6B infection can cause several clinical manifestations in transplant recipients including encephalitis, bone marrow suppression, and pneumonitis. In contrast to HHV-6B, the clinical features of HHV-6A infection remain largely undefined. Herein, we developed a multiplex cycling probe real-time PCR that discriminated between HHV-6A and HHV-6B. The assay was HHV-6-specific and no cross amplification was demonstrated for other herpesviruses. Moreover, the assay had a broad, linear dynamic range of detection between 1 and 106 copies of viral DNA. The quantification of HHV-6A DNA was suppressed by an excess amount of HHV-6B DNA (1 × 106 copies/tube) in the multiplex PCR assay; however, 1 × 106 copies/tube of HHV-6A DNA did not affect the quantification of 1 × 104 copies/tube of HHV-6B DNA. To determine the reliability of the assay for analysis of clinical specimens, DNAs extracted from the peripheral blood of hematopoietic stem cell transplant recipients were assayed using our multiplex real-time PCR versus the standard TaqMan PCR. Strong correlations were demonstrated between the two different assay systems for both HHV-6A (R2 = 0.913) and HHV-6B (R2 = 0.909). Therefore, our multiplex HHV-6 species-specific cycling probe real-time PCR is useful for evaluating the precise copy numbers of HHV-6A and B in transplant recipients. However, as HHV-6A copy numbers was affected by presence of high copies of HHV-6B DNA (1 × 106 copies/tube), it may be difficult to measure precise copy numbers of HHV-6A in inherited chromosomally integrated HHV-6B patient. J. Med. Virol. 88:1628–1635, 2016.

AB - Human herpesvirus 6 (HHV-6) is classified as two distinct species: HHV-6A and B. HHV-6B infection can cause several clinical manifestations in transplant recipients including encephalitis, bone marrow suppression, and pneumonitis. In contrast to HHV-6B, the clinical features of HHV-6A infection remain largely undefined. Herein, we developed a multiplex cycling probe real-time PCR that discriminated between HHV-6A and HHV-6B. The assay was HHV-6-specific and no cross amplification was demonstrated for other herpesviruses. Moreover, the assay had a broad, linear dynamic range of detection between 1 and 106 copies of viral DNA. The quantification of HHV-6A DNA was suppressed by an excess amount of HHV-6B DNA (1 × 106 copies/tube) in the multiplex PCR assay; however, 1 × 106 copies/tube of HHV-6A DNA did not affect the quantification of 1 × 104 copies/tube of HHV-6B DNA. To determine the reliability of the assay for analysis of clinical specimens, DNAs extracted from the peripheral blood of hematopoietic stem cell transplant recipients were assayed using our multiplex real-time PCR versus the standard TaqMan PCR. Strong correlations were demonstrated between the two different assay systems for both HHV-6A (R2 = 0.913) and HHV-6B (R2 = 0.909). Therefore, our multiplex HHV-6 species-specific cycling probe real-time PCR is useful for evaluating the precise copy numbers of HHV-6A and B in transplant recipients. However, as HHV-6A copy numbers was affected by presence of high copies of HHV-6B DNA (1 × 106 copies/tube), it may be difficult to measure precise copy numbers of HHV-6A in inherited chromosomally integrated HHV-6B patient. J. Med. Virol. 88:1628–1635, 2016.

UR - http://www.scopus.com/inward/record.url?scp=84977659298&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84977659298&partnerID=8YFLogxK

U2 - 10.1002/jmv.24513

DO - 10.1002/jmv.24513

M3 - Article

C2 - 26945690

AN - SCOPUS:84977659298

VL - 88

SP - 1628

EP - 1635

JO - Journal of Medical Virology

JF - Journal of Medical Virology

SN - 0146-6615

IS - 9

ER -