METHOD AND SET FOR RAPID PERTUSSIS DIAGNOSIS Russian patent published in 2009 - IPC C12Q1/68 C12N15/10 

FIELD: medicine.

SUBSTANCE: invention refers to medical microbiology, specifically to laboratory diagnostics of pertussis agent. Method provides that clinical material is sampled from oral pharynx of the patient, with DNA separation, polymerase chain reaction and electrophoresis. Herewith clinical material is consistently warmed up at 95°C during 30 min, processed with lysozyme at 37°C±2°C during 30 min and proteinase K at 70°C within 2 hours. DNA separation is enabled through processing with lysing reagent and incubation at 65°C during 5 min, thereafter processing with sorbent at room temperature during 7 min, wash solution and elution with TE-buffer at 65°C within 5 minutes. PCR is performed with PCR reaction mixture, containing 10×PCR buffer with (NH₄)₂SO₄, 2mM mixed nucleotides (dNTPs), 25mM MgCl₂, 5% DMSO, three primer pairs BP-F3 5'-CCGCATACGTGTTGGCA-3' BP-B3 5'-TGCGTTTTGATGGTGCCT-3' BP-FIP5'- TTGGATTGC AGTAGCGGGATGTGC ATGCGTGCAGATTCGTC-3', BP-BIP5'-CGCAAAGTCGCGCGATGGTAACGGATCACACCATGGCA-3', BP-LF 5'-ACGGAAGAATCGAGGGTTTTGTAC-3' BP-LB 5'- GTCACCGTCCGGACCGTG-3' and Bst polymerase. Herewith amplification is cycling, at first at 65°C during 60 min, then at 80°C during 2 min, thereafter the cycle is repeated. After electrophoresis, PCR products are differentiated by comparing electrophoretic mobility of produced DNA fragments and mobility of DNA check samples of strains Bordetella pertussis. If specific luminous profile similar to check sample is found, DNA sample is recorded to contain DNA of strain B.pertussis, thus pertussis is diagnosed. Method is ensured by application of the set containing reagents listed above and check sample of DNA B.pertussis.

EFFECT: possibility to reveal disease agent within 9-10 hours from analysis beginning directly in the material from the patient that reduces diagnosis time, prescribing timely and adequate therapy.

2 cl, 2 tbl, 1 ex