Validation of the suppressive subtractive hybridization method in Mycoplasma agalactiae species by the comparison of a field strain with the type strain PG2Reportar como inadecuado




Validation of the suppressive subtractive hybridization method in Mycoplasma agalactiae species by the comparison of a field strain with the type strain PG2 - Descarga este documento en PDF. Documentación en PDF para descargar gratis. Disponible también para leer online.



Abstract : The subtractive suppressive hybridization SSH, a method that allows the identification of sequences that are present in one genome tester but not in the other driver, is a promising technique for the comparison of Mycoplasma agalactiae pathogenic strains. The optimal conditions for SSH were established by subtracting the M. agalactiae type strain PG2 DNA from the M. agalactiae strain 5632 DNA. Because these two strains possess different vpma gene repertoires, 5632-specific vpma sequences and possibly other 5632-specific sequences were predicted to be retrieved by SSH. The subtracted tester DNA was PCR-amplified and cloned into the pGEM-T easy E. coli vector. Two independent libraries were generated and used to prepare individual probes that were tested by Southern blot with genomic DNA from various field isolates and mycoplasma reference strains. Sequence analysis of two overlapping clones showed that they potentially code for a large carboxyterminal portion of a new vpma ORF. Several DNA fragments homologous to insertion sequences were also found in 5632 and related strains. These preliminary data suggest that SSH is a powerful method to investigate differences between mycoplasma strains, and may be applied to molecular epidemiology, diagnostic, and host specificity or pathogenicity determinant discovery.

Keywords : Mycoplasma agalactiae subtractive hybridization vpma insertion sequence





Autor: Marc Marenda Edy Vilei François Poumarat Joachim Frey Xavier Berthelot

Fuente: https://hal.archives-ouvertes.fr/



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