Laser-Raman spectroscopy: a novel approach for sensitive molecular characterization of the mycobacterium genomic DNAs

In the present work we give a detailed analysis on the key Raman markers of genomic DNA for four species of Mycobacterium namely: Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium avium and Mycobacterium phlei. The fundamental groups of bands have done for the first time to these Mycobacterium genomic DNAs. Molecular Laser Raman Spectroscopy [MLRS]; a novel and highly sensitive method, was used for whole genome characterization and comparison of the four species under investigation. The Raman spectroscopic analysis showed, in the M. avium and M. bovis the DNA is more rich in A+T as compared with the others, while the M. phlei and M tuberculosis are more rich in G+C. Moreover, a detailed analysis of all the bands diagnostic of B-form backbone geometry; the phosphate groups, bands diagnostic of deoxynucleoside conformation, ring vibrations of the bases, carbonyl stretching modes of thymine, guanine and cytosine was given here for the four mycobacterial species DNAs. The present study describes the use of [MLRS], as a novel promising assay, for rapid and discriminative detection of a mycobacterial genome with no confusion with other bacteria, utilizing genomes of four different mycobacteria species. In addition, Molecular Polymorphic Laser Raman Spectroscopy fingerprints and / or sequences may serve as markers that provide a basis for virulence, phylogenetic and evolutionary studies

Genetic characterization of the Egyptian vaccinal strain [Abu-Hammad] of bovine herpesvirus-1

The local Egyptian vaccinal strain [Abu-Hammad] of bovine herpesvirus-I [BHV-1] was genetically characterized based on two main molecular approaches; the Hind III endonuclease for genomic fingerprinting of the local BHV-1 compared to that of the reference strain [Cooper 1] of BHV-1 subtype 1 [BH V-1.1], and the analyses of nucleotide [nt] sequences, deduced amino acid [N sequences and phylogeny of the major viral immunogen, glycoprotein D [gD] of the local BHVmsu: its counterparts of other related alpha-herpesviruses. The resulted sizes and c‘iccaophoretic patterns of the Hind III viral DNA fragments [A -to-M] revealed close identity between the local BHV-1 and reference BHV-1.1. Both nt and deduced aa sequence alignments revealed variable degrees of similarity of the local BHV-1 gD, which was high with BHV-1.1 and BHV-1.2; moderate with bovine herpesvrius-5; low with caprine herpesvirus-1 and suid herpesvirus-1 [pseudorabies virus]; or very low with human herpesvirus-1 and herpesvirus-2. A possible mutational frame shift at nt 509 and 615 was observed toward the carboxyl-terminus of the local BHV-1 gD. The gD nt and deduced aa sequence data enabled phylogenetic characterization of the local BHV-1 and correlated with the results of genomic fingerprinting after restriction endonuclease cleavage. Based on phylogenetic analyses, the Egyptian vaccinal BHV-1 was grouped as a BHV-1 subtype 1 in a distinguished branch within the phylogenetic tree, together with BHV1.1. Determined conservation offive cysteine residues and the glycosylation domains in the amino [N-] terminal half emphasized the importance of the N-terminus for immunological and biological function of gD among alpha-herpesviruses. Presence of the most divergent domain of 17 aa residues at positions 168-184 and an additional cysteine residue at position 178 could be used as tools to distinguish the local Egyptian BHV-1 from other related herpesviruses. Findings of this work showed that genomic fingerprinting, based on endonuclease Hind III cleavage, and direct sequencing of the gD gene-derived PCR amplicons were relevant tools for genetic characterization of BHV-1 strains / isolates. The comparative genetic analyses conducted were useful to trace conservation of the local BHV-1 among related alpha-herpesviruses and to establish genetic tools for national-wide epidemiological studies and development of novel efficient BHV-1 vaccines