Development of quantitative RT-PCR assays for detection of three classes of HHV-6B gene transcripts

Masaru Ihira, Yoshihiko Enomoto, Yoshiki Kawamura, Hidetaka Nakai, Ken Sugata, Yoshizo Asano, Motohiro Tsuzuki, Nobuhiko Emi, Tatsunori Goto, Koichi Miyamura, Kimikazu Matsumoto, Koji Kato, Yoshiyuki Takahashi, Seiji Kojima, Tetsushi Yoshikawa

Research output: Contribution to journalArticle

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Abstract

The monitoring of active human herpesvirus 6 (HHV-6) B infection is important for distinguishing between the reactivation and latent state of the virus. The aim of this present study is to develop a quantitative reverse transcription polymerase chain reaction (RT-PCR) assay for diagnosis of active viral infection. Primers and probes for in house quantitative RT-PCR methods were designed to detect the three kinetic classes of HHV-6B mRNAs (U90, U12, U100). Stored PBMCs samples collected from 10 patients with exanthem subitum (primary HHV-6B infection) and 15 hematopoietic stem cell transplant recipients with HHV-6B reactivation were used to evaluate reliability for testing clinical samples. Excellent linearity was obtained with high correlation efficiency between the diluted RNA (1-100ng/reaction) and Ct value of each gene transcript. The U90 and U12 gene transcripts were detected in all of the peripheral blood mononuclear cells (PBMCs) samples collected in acute period of primary HHV-6B infection. Only one convalescent PBMCs sample was positive for the U90 gene transcript. Additionally, the reliability of HHV-6B quantitative RT-PCRs for diagnosis of viral reactivation in hematopoietic transplant recipients was evaluated. Relative to virus culture, U90 quantitative RT-PCR demonstrated the highest assay sensitivity, specificity, positive predictive value, and negative predictive value. Thus, this method could be a rapid and lower cost alternative to virus culture, which is difficult to perform generally, for identifying active HHV-6B infection. J. Med. Virol. 84:1388-1395, 2012. © 2012 Wiley Periodicals, Inc.

Original languageEnglish
Pages (from-to)1388-1395
Number of pages8
JournalJournal of Medical Virology
Volume84
Issue number9
DOIs
Publication statusPublished - 01-09-2012

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Human Herpesvirus 6
Reverse Transcription
Polymerase Chain Reaction
Genes
Blood Cells
Viruses
Infection
Cercopithecine Herpesvirus 1
Virus Diseases
Hematopoietic Stem Cells
Exanthema
RNA
Transplants
Costs and Cost Analysis
Sensitivity and Specificity
Messenger RNA

All Science Journal Classification (ASJC) codes

  • Infectious Diseases
  • Virology

Cite this

Ihira, Masaru ; Enomoto, Yoshihiko ; Kawamura, Yoshiki ; Nakai, Hidetaka ; Sugata, Ken ; Asano, Yoshizo ; Tsuzuki, Motohiro ; Emi, Nobuhiko ; Goto, Tatsunori ; Miyamura, Koichi ; Matsumoto, Kimikazu ; Kato, Koji ; Takahashi, Yoshiyuki ; Kojima, Seiji ; Yoshikawa, Tetsushi. / Development of quantitative RT-PCR assays for detection of three classes of HHV-6B gene transcripts. In: Journal of Medical Virology. 2012 ; Vol. 84, No. 9. pp. 1388-1395.
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abstract = "The monitoring of active human herpesvirus 6 (HHV-6) B infection is important for distinguishing between the reactivation and latent state of the virus. The aim of this present study is to develop a quantitative reverse transcription polymerase chain reaction (RT-PCR) assay for diagnosis of active viral infection. Primers and probes for in house quantitative RT-PCR methods were designed to detect the three kinetic classes of HHV-6B mRNAs (U90, U12, U100). Stored PBMCs samples collected from 10 patients with exanthem subitum (primary HHV-6B infection) and 15 hematopoietic stem cell transplant recipients with HHV-6B reactivation were used to evaluate reliability for testing clinical samples. Excellent linearity was obtained with high correlation efficiency between the diluted RNA (1-100ng/reaction) and Ct value of each gene transcript. The U90 and U12 gene transcripts were detected in all of the peripheral blood mononuclear cells (PBMCs) samples collected in acute period of primary HHV-6B infection. Only one convalescent PBMCs sample was positive for the U90 gene transcript. Additionally, the reliability of HHV-6B quantitative RT-PCRs for diagnosis of viral reactivation in hematopoietic transplant recipients was evaluated. Relative to virus culture, U90 quantitative RT-PCR demonstrated the highest assay sensitivity, specificity, positive predictive value, and negative predictive value. Thus, this method could be a rapid and lower cost alternative to virus culture, which is difficult to perform generally, for identifying active HHV-6B infection. J. Med. Virol. 84:1388-1395, 2012. {\circledC} 2012 Wiley Periodicals, Inc.",
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Ihira, M, Enomoto, Y, Kawamura, Y, Nakai, H, Sugata, K, Asano, Y, Tsuzuki, M, Emi, N, Goto, T, Miyamura, K, Matsumoto, K, Kato, K, Takahashi, Y, Kojima, S & Yoshikawa, T 2012, 'Development of quantitative RT-PCR assays for detection of three classes of HHV-6B gene transcripts', Journal of Medical Virology, vol. 84, no. 9, pp. 1388-1395. https://doi.org/10.1002/jmv.23350

Development of quantitative RT-PCR assays for detection of three classes of HHV-6B gene transcripts. / Ihira, Masaru; Enomoto, Yoshihiko; Kawamura, Yoshiki; Nakai, Hidetaka; Sugata, Ken; Asano, Yoshizo; Tsuzuki, Motohiro; Emi, Nobuhiko; Goto, Tatsunori; Miyamura, Koichi; Matsumoto, Kimikazu; Kato, Koji; Takahashi, Yoshiyuki; Kojima, Seiji; Yoshikawa, Tetsushi.

In: Journal of Medical Virology, Vol. 84, No. 9, 01.09.2012, p. 1388-1395.

Research output: Contribution to journalArticle

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T1 - Development of quantitative RT-PCR assays for detection of three classes of HHV-6B gene transcripts

AU - Ihira, Masaru

AU - Enomoto, Yoshihiko

AU - Kawamura, Yoshiki

AU - Nakai, Hidetaka

AU - Sugata, Ken

AU - Asano, Yoshizo

AU - Tsuzuki, Motohiro

AU - Emi, Nobuhiko

AU - Goto, Tatsunori

AU - Miyamura, Koichi

AU - Matsumoto, Kimikazu

AU - Kato, Koji

AU - Takahashi, Yoshiyuki

AU - Kojima, Seiji

AU - Yoshikawa, Tetsushi

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