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J Med Microbiol 52 (2003), 793-799; DOI: 10.1099/jmm.0.05192-0
© 2003 Society for General Microbiology
ISSN 0022-2615

Direct-test PCR for detection of meningococcal DNA and its serogroup characterization: standardization and adaptation for use in a public health laboratory

L. F. Baethgen1,2, C. Moraes2, L. Weidlich2, S. Rios3, C. Í. Kmetzsch4, M. S.N. Silva2, M. L.R. Rossetti2 and A. Zaha1

1Centro de Biotecnologia do Estado do Rio Grande do Sul, Universidade Federal do Rio Grande do Sul, Campus do Vale, Caixa Postal 15.005, CEP: 91.501-970 Porto Alegre, RS, Brazil 2Centro de Desenvolvimento Científico e Tecnológico da Fundação Estadual de Produção e Pesquisa em Saúde (CDCT/FEPPS), Porto Alegre, RS, Brazil 3Instituto de Pesquisas Biológicas – Laboratório Central de Saúde Pública do Estado do Rio Grande do Sul (IPB-LACEN/RS), Porto Alegre, RS, Brazil 4Divisão de Controle de Doenças Transmissíveis Agudas, Setor de Epidemiologia, Secretaria da Saúde, Porto Alegre, RS, Brazil

Correspondence A. Zaha zaha{at}dna.cbiot.ufrgs.br

Received January 24, 2003
Accepted May 8, 2003

A direct PCR test (DT-PCR) was established to detect Neisseria meningitidis DNA in clinical samples from patients with suspected bacterial meningitis. Specific primers for the 16S rDNA of N. meningitidis were designed to amplify a 600 bp DNA fragment. One hundred and ninety-three clinical samples were analysed, corresponding to 114 samples from patients diagnosed as positive and 79 as negative for infection by N. meningitidis using conventional methods (culture, latex agglutination and counterimmunoelectrophoresis). These samples were submitted to PCR by two different clinical sample preparation approaches (with and without DNA extraction and purification) and submitted to different PCR protocols to improve the results. In agarose gel detection, the sensitivity value for DT-PCR was 88.5 % and, using dot-blot DNA detection, the sensitivity increased to 96.4 %. The detection limit for meningococcus in cerebrospinal fluid was 2x102 c.f.u. ml-1. Serogroup prediction was done using a multiplex PCR protocol and the sensitivity was 83 % for agarose gel DNA detection and 96.4 % using dot-blot DNA detection.

Abbreviations: CIE, counterimmunoelectrophoresis; CSF, cerebrospinal fluid; DT-PCR, direct-test PCR; GM-PCR, glass-matrix PCR; LA, latex agglutination.






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