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Borisova, Anastasia Borisovna; Urban, Yu N; Gadua, N T; Borisova, O Yu; Pimenova, A S; Afanasiev, M S; Petrova, M S; Afanasiev, S S; Smetanina, S V
Klinicheskaia laboratornaia diagnostika, 2020-Sep-16, Letnik: 65, Številka: 9Journal Article
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. 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. 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. 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.
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JCR | SNIP | JCR | SNIP | JCR | SNIP | JCR | SNIP |
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