The application of mass spectrometry method for the study and identification of medically important viruses (review of literature).

It is difficult to overestimate the urgency of the problem of well-timed diagnosis of viral infections. According to the WHO, dozens of outbreaks of viral diseases are recorded annually, both in developing and developed countries. Moreover, the seasonal flu virus alone is capable of infecting up to 20% of the population, even in European countries with a high level of medicine. And the annual number of deaths due to viral infections, according to official statistics, exceeds 600 thousand people around the world. That's why the provision of a reliable and fairly rapid diagnosis of viruses, along with subsequent therapy, makes a significant contribution to reducing the incidence of mortality. Despite the fact that PCR-based methods currently remain the most common method for identifying viruses in clinical practice, as recent experience shows, in addition to the already known disadvantages, in the event of large outbreaks, such test systems may simply not be in the required amount. In this regard, it is necessary to supplement and improve the existing tools for identification and research of clinically significant viruses. The MALDI-TOF mass spectrometry method combines a degree of accuracy and versatility, sufficient both for the identification of clinical strains isolated from patients, and for the study of the phenotypic properties of viruses in research laboratories and centers. This article presents and summarizes the main data on the existing or potential application of the method of time-of-flight mass spectrometry with matrix-associated laser desorption / ionization for the identification or study of viruses.

Modification of the Method of Receiving of Insertion Mutants with the EZ::TN5 System.

We demonstrated the possibility of obtaining insertion mutants by a modified technique using EZ::TN5 system during culturing of the recipient strain on a dense nutrient medium and exclusion of the centrifugation stage. The frequency of transposon mutants of E. coli 10979/EZ::TN5 was 2×10-6. Genetically modified strains were characterized by kanamycin resistance, inability to L-malate assimilation, changes in the expression of individual proteins of protein mass-spectra (5096.3, 6252.9, and 9067.7 Da), and the presence of fragments in genomic DNA amplified by specific forward and reverse primers that were homologous to Tn5 transposon insertion sites. The modified procedure for obtaining insertion mutants by using EZ::TN5 system was not inferior by the efficiency to the standard procedure, but shortens experiment duration, simplifies it, and reduces the risks related to working with group 2 pathogenicity microorganisms.

[The problems of identification of various strains of vegetative form of Bacillus anthracis using MALDI-ToF MS technique.]

The article considers experience of application of mass-spectrometry with matrix-activated laser desorption/ionization for fast and reliable identification of Bacillus anthracis and heterologous species of microorganisms. The particular interesting characteristics and difficulties occurred during identification are covered.

[The application of time-of-flight mass spectrometry with matrix activated laser desorption-ionization (MALDI-ToF) for identifying agents of glanders and melioidosis].

The article presents the results of application of developed methodological approach to identifying Burkholderia pseudomallei and Burkholderia mallei using direct mass spectrometry profiling of cellular proteins. The protocol of sampling preparation of cultures of melioidosis and glanders was optimized with taking in account characteristics of observation of requirements of biological safety for operations with pathogenic biological agents of pathogenicity group II. The dependence of quality of mass spectrums (number of individual peaks and their intensity) from medium of fermentation of microorganisms was evaluated. The characteristic mass spectrums of collection strains B.pseudomallei (5) and B.mallei (5) were obtained. The set of reference mass-spectrums was generated for identification data base S.A.R.A.M.I.S.TM (Anagnostec Gmbh.). The mentioned data base was used for identification of 43 strains of pathogenic Burkholderia. The opportunity of reliable identification of taxonomic belonging of examined microorganisms up to species' level. The cluster analysis of obtained mass-spectrums of common cellular proteins of collection strains of pathogenic Burkholderia demonstrated grouping of examined strains according to their species' belonging. The supplemented data base of mass-spectral characteristics hereinafter will permit applying express-identification of isolates suspicious for belonging to agents of melioidosis and glanders. The updated data base will become a basis for developing schemes of hemotyping of strains of Burkholderia using mass spectrometry technique.