[Real-time pcr procedure for determination of Streptococcus pneumoniae serotypes].

AIM: Development and application of real-time PCR (RT-PCR) procedure for determination of Streptococcus pneumoniae serotypes. MATERIALS AND METHODS: S. pneumoniae cps-locus wzx, wzy, wzz, wcwV and galU genes were chosen as PCR targets to select serotype-specific oligonucleotide primers and fluorescent labeled probes. 89 samples of cerebrospinal fluid (CSF) obtained in 2007 - 2010 from patients with pneumococcal meningitis diagnosis undergoing therapy in the Infectious Clinical Hospital No. 2, Moscow, were studied with the aim of testing the possibility of practical use of RT-PCR. RESULTS: Primers and probes were selected for the determination of 16 vaccine and/or frequently encountered serotypes distributed among 4 reaction mixtures also including a pair of primers and a probe for cpsA gene detection that is present in all the capsule pneumococci (internal control). The procedure was tested on a collection of 108 pneumococci strains gathered in Research Institute of Antimicrobial Therapy and serotyped earlier by specific PCR with electrophoretic detection and serologically by using Pneumotest-Latex kit. The sensitivity and specificity of the RT-PCR was 100%. RT-PCR procedure allowed to determine pneumococcus serotype in 79% of CSF clinical samples containing S. pneumoniae DNA. Serotype 3 and 23F were detected most frequently (13%, each). CONCLUSION: RT-PCR application does not assume causative agent seeding stage, significantly reduces analysis execution time and increases sensitivity of the study. The developed procedure will allow to begin addressing the important problem--clarification of spectra and frequency of occurrence of pneumococci serotypes circulating on the territory of Russia.

[A method for determination of Neisseria meningitidis serogroup A, B, C and W by real-time PCR].

AIM: Development and testing of a real-time PCR method for detection of Neisseria meningitiis serogroup A, B, C and W DNA. MATERIALS AND METHODS: Reference strains and 187 samples of cerebrospinal fluid (CSF) from meningocci meningitis patients were used in the study. Multiplex PCR was carried out in an instrument with 5 channels of fluorescent detection: RESULTS: Analysis of specific serogroup loci of the genome and design of oligonucleotides for the detection of DNA of all the capsule meningococci and 4 serogroups in particular was carried out. PCR conditions were optimized; specificity was shown and analytical sensitivity was evaluated using reference strains. DNA of the following serogroups was detected during study of clinical CSF samples: A--in 103 samples (55%), B--in 45 (24%), C--in 30 (16%), W--in 5 (3%). Only DNA of meningococci capstle gene ctrA was found in 4 samples; presumably, they contained DNA of other serogroups. Multilocus sequence-typing and detection of antigenic determinants of PorA and FetA genes for 27 DNA samples of group A menincococci as well as DNA of 5 group W meningococci and 4 ungroupable was carried out. CONCLUSION: The method proposed allows to carry out serogrouping of no less than 95% of strains or DNA samples isolated from CSF of meningococci infection patients. Combined with other recommended non-cultural methods of genotyping, it may be useful for complex characteristics of pathogenic meningococci.