Development of accelerated genodiagnosis method of pertussis and pertussis-like diseases on the basis of mPCR-RT.

ABSTRACT

The aim of the work was to develop an accelerated genodiagnosis method based on mPCR-RT for the detection DNA of B. pertussis, B. parapertussis, B. holmesii. MATERIALS AND METHODS: The study used 104 strains of microorganisms, of which 50 strains of B. pertussis, 37 - B. parapertussis, 17 - heterologous species of microorganisms. Assessment of analytical specificity was carried out using DNA strains of various microorganisms with a concentration at least 109 GE / ml. To check the analytical sensitivity we studied a series of serial dilutions of bacterial cultures of the control strains B. pertussis № 143, B. parapertussis № 38b, B. holmesii DSM 13416 with a concentration of 5x109 - 5 µm/ml. RESULTS: Insertion sequences were chosen as diagnostic targets for B. parapertussis - a specific fragment IS1001, for B. holmesii - a specific fragment hlIS1001, for B.pertussis - a fragment IS481. To develop a genodiagnosis method specific primers were designed and combined into a single multi-primer mixture, the composition of the reaction mixture and the amplification conditions were selected. The analytical sensitivity of the developed method for detecting pertussis and pertussis-like pathogens was 5×101 GE / ml. Verification of the developed methodology of gene diagnostics showed 100% analytical specificity. CONCLUSION: An accelerated genodiagnosis method based on mPCR-RT has been developed, it allows you to identify DNA of B. pertussis, B. parapertussis, B. holmesii, which expands the possibilities of examining patients with suspected pertussis and pertussis-like diseases in order to increase laboratory confirmation of the diagnosis.