Use of control materials designed for PCR diagnostics in assessing quality of bacterial detection by NGS sequencing of the 16S rRNA gene

Objective. To assess quality of bacterial detection using 16S rRNA gene sequencing with accredited control materials designed for external quality control of PCR studies. Materials and Methods. Control materials obtained as part of the ISI «FSVOK» program: «DNA detection of Mycoplasma hominis, Ureaplasma spp., Ureaplasma urealyticum, Ureaplasma parvum by PCR (STI)», were analyzed by 16S rRNA gene sequencing. The control sets of samples were presented with lyophilized solutions of M. hominis, U. urealyticum, and U. parvum genomic DNA at specified concentrations and human genomic DNA at a concentration of at least 2 × 105 copies/ml in two cycles. Results. We analyzed 16 control samples that were obtained in two MCI cycles. The analysis of control materials presented in the MCI program «FSVOK» using 16S rRNA gene sequencing allowed for an accurate qualitative assessment of the presence or absence of opportunistic microorganisms such as M. hominis and Ureaplasma spp. A limitation of the method is the difficulty in species identification of Ureaplasma parvum/urealyticum due to the similarity in nucleotide sequences in the V3-V4 region of the 16S rRNA. Despite the very high sensitivity of the method and the detection of other microorganisms at extremely low values (< 0.1%), the negative control sample, which did not contain the target bacterial markers, did not reveal M. hominis or Ureaplasma spp in two cycles of the program, indicating the absence of contamination at all stages of laboratory research, OPK production, storage, and distribution by the MCI organizer, as well as the absence of false-positive results in the application of the 16S rRNA gene sequencing method. Conclusions. The method allows reliable identification of M. hominis, Ureaplasma spp., U. urealyticum, U. parvum, and other bacteria in control samples, which may have clinical significance in assessing vaginal microbiocenosis, as an advantage over the PCR method designed to detect specific individual targets.