Development of an assay for the detection and quantification of the measles virus nucleoprotein (N) gene using real-time reverse transcriptase PCR.
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 10(1)-10(7) copies per reaction (corresponding to 5x10(-1)-5x10(5) copies microl(-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, 3.9x10(3)-5.2x10(6) copies ml(-1) and 7.4x10(7)-2.0x10(8) 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.
Akiyama
Miho
M
Infectious Disease Surveillance Center, National Institute of Infectious Diseases, 4-7-1 Gakuen, Musashimurayama, Tokyo 208-0011, Japan.
Kimura
Hirokazu
H
Infectious Disease Surveillance Center, National Institute of Infectious Diseases, 4-7-1 Gakuen, Musashimurayama, Tokyo 208-0011, Japan.
Tsukagoshi
Hiroyuki
H
Gunma Prefectural Institute of Public Health and Environmental Sciences, 378 Kamioki-machi, Maebashi, Gunma 371-0052, Japan.
Taira
Katsuya
K
Okinawa Prefectural Institute of Public Health and Environmental Sciences, 2085 Ozato, Nanjo, Okinawa 910-1202, Japan.
Mizuta
Katsumi
K
Yamagata Prefectural Institute of Public Health, 1-6-6 Tokamachi, Yamagata 990-0031, Japan.
Saitoh
Mika
M
Gunma Prefectural Institute of Public Health and Environmental Sciences, 378 Kamioki-machi, Maebashi, Gunma 371-0052, Japan.
Nagano
Manami
M
Molecular and Cellular Biology Division, Applied Biosystems Japan Ltd, 4-5-4 Hatchobori, Chuo-ku, Tokyo 104-0032, Japan.
Sutoh
Asuka
A
Yamagata Prefectural Institute of Public Health, 1-6-6 Tokamachi, Yamagata 990-0031, Japan.
Noda
Masahiro
M
Department of Virology III, National Institute of Infectious Diseases, 4-7-1 Gakuen, Musashimurayama, Tokyo 208-0011, Japan.
Morita
Yukio
Y
Gunma Prefectural Institute of Public Health and Environmental Sciences, 378 Kamioki-machi, Maebashi, Gunma 371-0052, Japan.
Sakatsume
Osamu
O
Molecular and Cellular Biology Division, Applied Biosystems Japan Ltd, 4-5-4 Hatchobori, Chuo-ku, Tokyo 104-0032, Japan.
Okabe
Nobuhiko
N
Infectious Disease Surveillance Center, National Institute of Infectious Diseases, 4-7-1 Gakuen, Musashimurayama, Tokyo 208-0011, Japan.
Tashiro
Masato
M
Department of Virology III, National Institute of Infectious Diseases, 4-7-1 Gakuen, Musashimurayama, Tokyo 208-0011, Japan.
D017931:DNA Primers; D005784:Gene Amplification; D005814:Genes, Viral / Q000235:genetics; D005838:Genotype; D006801:Humans; D008457:Measles / Q000821:virology*; D008459:Measles virus / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D019590:Nucleocapsid Proteins / Q000235:genetics*; D010614:Pharynx / Q000821:virology; D010957:Plasmids; D012367:RNA, Viral / Q000235:genetics / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*
D017931:DNA Primers; D019590:Nucleocapsid Proteins; D012367:RNA, Viral