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Measles Virus Genotyping by Nucleotide-Specific Multiplex PCR

Jacques R. Kremer,^1,2 Fred Fack,¹ Christophe M. Olinger,¹ Mick N. Mulders,^1, and Claude P. Muller^1,2*

Department of Immunology and WHO Collaborative Centre for Measles, Laboratoire National de Santé, L-1011 Luxembourg, Luxembourg,¹ Ecole doctorale BioSE, Université Henri Poincaré, F-54505 Vandoeuvre-lès-Nancy Cedex, France²

Received 3 February 2004/ Returned for modification 26 February 2004/ Accepted 4 April 2004

A simple genotyping method based on multiplex PCR has been developed to discriminate between all active measles virus (MV) clades and genotypes (A, B3.1, B3.2, C2, D2-D9, G2-G3, and H1-H2). The sequencing reaction was replaced by six multiplex PCRs: one to identify the clade and five to identify the respective genotype. Primers were sensitive to clade- and genotype-specific nucleotides and generated fragments of type-specific sizes that were analyzed by conventional agarose gel electrophoresis. On the basis of all published MV sequences, positive and negative predictive values of 99.2% and 98.6% were calculated. Variability in the primer binding sites, which could potentially reduce sensitivity, was very limited among published sequences. As new genotypes are described, additional specific primers can be included in the multiplex PCR with relative ease. Although sequencing remains the "gold standard," the present method should facilitate MV genotyping especially in developing countries and will therefore contribute to enhanced MV control and elimination strategies as recommended by the World Health Organization.

Present address: WHO European Regional Office, Copenhagen, Denmark.

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