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Development of an assay for the detection and quantification of the measles virus nucleoprotein (N) gene using real-time reverse transcriptase PCR

Miho Akiyama^1,, Hirokazu Kimura^1,, Hiroyuki Tsukagoshi², Katsuya Taira³, Katsumi Mizuta⁴, Mika Saitoh², Manami Nagano⁵, Asuka Sutoh⁴, Masahiro Noda⁶, Yukio Morita², Osamu Sakatsume⁵, Nobuhiko Okabe¹ and Masato Tashiro⁶

¹ Infectious Disease Surveillance Center, National Institute of Infectious Diseases, 4-7-1 Gakuen, Musashimurayama, Tokyo 208-0011, Japan

² Gunma Prefectural Institute of Public Health and Environmental Sciences, 378 Kamioki-machi, Maebashi, Gunma 371-0052, Japan

³ Okinawa Prefectural Institute of Public Health and Environmental Sciences, 2085 Ozato, Nanjo, Okinawa 910-1202, Japan

⁴ Yamagata Prefectural Institute of Public Health, 1-6-6 Tokamachi, Yamagata 990-0031, Japan

⁵ Molecular and Cellular Biology Division, Applied Biosystems Japan Ltd, 4-5-4 Hatchobori, Chuo-ku, Tokyo 104-0032, Japan

⁶ Department of Virology III, National Institute of Infectious Diseases, 4-7-1 Gakuen, Musashimurayama, Tokyo 208-0011, Japan

Correspondence
Hirokazu Kimura
kimhiro{at}nih.go.jp

Received July 31, 2008
Accepted January 18, 2009

We developed a new quantification method for the measles virus (MeV) nucleoprotein (N) gene using real-time reverse transcriptase PCR. This method allowed us to quantify 10¹–10⁷ copies per reaction (corresponding to 5x10^–1–5x10⁵ copies µl^–1) of the MeV N gene. We also quantified the MeV N gene from the throat swabs of 22 patients with measles as well as the MeV genotypes A, D3, D5, D9 and H1 in viral suspensions derived from MeV-infected cells. As a result, 3.9x10³–5.2x10⁶ copies ml^–1 and 7.4x10⁷–2.0x10⁸ copies ml^–1 of the MeV genomes (N gene) were detected in the throat swabs and viral suspensions, respectively. No other viruses (enteroviruses, respiratory syncytial virus, human metapneumovirus or mumps virus) were detected in the assay. The results suggest that this method is applicable to the detection and quantification of some genotypes of MeV.

Abbreviations: C_t, threshold cycle; RT-PCR, reverse transcriptase PCR.

These authors contributed equally to this work.

The GenBank/EMBL/DDBJ accession numbers for the measles virus nucleoprotein gene sequences are AB447494–AB447515.