Elsevier

Journal of Clinical Virology

Volume 80, July 2016, Pages 98-101
Journal of Clinical Virology

Short communication
Evaluation of sensitivity of TaqMan RT-PCR for rubella virus detection in clinical specimens

https://doi.org/10.1016/j.jcv.2016.05.005Get rights and content

Highlights

  • We evaluated a TaqMan RT-PCR assay for the rubella surveillance system in Japan.

  • We tested its sensitivity with all rubella virus genotypes and clinical specimens.

  • Detection limits for the rubella virus genotypes ranged from 1 to 10 pfu/reaction.

  • The sensitivity of the TaqMan RT-PCR was lower than the conventional nested RT-PCR.

  • The assay is appropriate for throat swab and urine samples until 5 days of illness.

Abstract

Background

An easy and reliable assay for detection of the rubella virus is required to strengthen rubella surveillance. Although a TaqMan RT-PCR assay for detection of the rubella virus has been established in Japan, its utility for diagnostic purposes has not been tested.

Objectives

To allow introduction of the TaqMan RT-PCR into the rubella surveillance system in Japan, the sensitivity of the assay was determined using representative strains for all genotypes and clinical specimens.

Study design

The detection limits of the method for individual genotypes were examined using viral RNA extracted from 13 representative strains. The assay was also tested at 10 prefectural laboratories in Japan, designated as local reference laboratories for measles and rubella, to allow nationwide application of the assay.

Results

The detection limits and amplification efficiencies of the assay were similar among all the representative strains of the 13 genotypes. The TaqMan RT-PCR could detect approximately 90% of throat swab and urine samples taken up to 5 days of illness. These samples were determined positive by a highly sensitive nested RT-PCR.

Conclusions

The TaqMan RT-PCR could detect at least 10 pfu of rubella virus. Although the sensitivity was somewhat lower than that of the conventional nested RT-PCR, the TaqMan RT-PCR could be more practical to routine tests for rubella laboratory diagnosis and detection in view of the rapid response and reducing risks of contamination.

Section snippets

Background

Rubella virus infection during early pregnancy often results in severe birth defects termed congenital rubella syndrome (CRS), which induces various symptoms, including sensorineural deafness, heart diseases, and cataracts [1], [2]. Although there are no specific treatments for rubella and CRS, efficient and safe live-attenuated vaccines are available for their prevention. In 2012, the World Health Assembly endorsed the Global Vaccine Action Plan 2011–2020 [3], which established elimination

Objectives

To introduce a previously reported TaqMan RT-PCR for RV genome detection into the nationwide surveillance system in Japan, its performance was evaluated using representative strains for all the RV genotypes and clinical specimens.

Sensitivity of the TaqMan RT-PCR for detection of all RV genotypes

RV reference strains for all 13 genotypes were used [9]. The viral genome was extracted from serial dilutions of each virus stock and subjected to the TaqMan RT-PCR as described previously [8], except that TaqMan Fast Virus 1-Step Master Mix (Life Technologies, Carlsbad, CA) was used as the reagent. The cDNA amplifications were conducted under conditions of 50 °C for 5 min, 95 °C for 20 s, and 45 cycles of 95 °C for 15 s and 60 °C for 1 min. Test samples were considered positive when amplification was

Results

To clarify the sensitivity for all RV genotypes, RNA from serially diluted representative strains of all the genotypes were subjected to the TaqMan RT-PCR. The assay was able to detect the RNA from 1 or 10 pfu of the representative strains (Table 2). In addition, the amplification of viral RNA from the representative strains showed comparable efficiency, ranging from 87.2 ± 0.05% to 102.0 ± 0.04%.

The results of the nested RT-PCR and TaqMan RT-PCR by types of clinical specimens are shown in Table 3.

Discussion

RV is genetically classified into 13 genotypes [10]. The primer and probe set for the TaqMan RT-PCR were designed to target the 5′-terminal p150-coding region. This nucleotide sequence is highly conserved among RV strains with various genotypes [11]. In fact, the present study indicates that the assay was able to detect all of the representative strains with comparable amplification efficiency. The genotyping analyses of the RV-positive clinical samples by the TaqMan RT-PCR in this study

Ethical approval

This project received ethical approval from the Research and Ethical Committees for the Use of Human Subjects of the National Institute of Infectious Diseases, Tokyo, Japan (No. 435).

Acknowledgements

We thank Drs. J.P. Icenogle, P. Rota, and all members of the Measles, Mumps, Rubella and Herpes Virus Laboratory Branch, Division of Viral Diseases, Centers for Disease Control and Prevention for the kind gifts of the RV representative strains. We also thank Ms. Yoko Ozaki, Osaka Prefectural Institute of Public Health. We wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could

References (14)

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