Development of cPCR technique for detection and enumeration of Prevotella bryantii ; Priprava metode cPCR za hitro odkrivanje in številčno vrednotenje celic bakterije Prevotella bryantii

Competitive PCR (cPCR) system for the detection and enumeration ofPrevotella bryantiicells in rumen samples was developed. PBB14 primer, specific forP. bryantiiwas used as the reverse PCR primer and EUB 338 bacterial primer as the forward primer. An internal DNA control, containing primer specific sequences at 5’ and 3’ ends, but lacking 41 bp at the middle of the sequence, was constructed. C-PCR products were quantified by capillary electrophoresis and the results were calculated with regression equation (Reilly and Attwood, 1998). By coamplification ofP.bryantiiB14 genomic DNA extracted from known numbers of cells and internal control, standard curve was constructed which enables quantification ofP.bryantiicells in the range of 2.15 x 103to 4.3 x 104cells. ; Razvili smo metodo kompetitivne PCR za odkrivanje in številčno vrednotenje bakterijskih celicPrevotella bryantiiv vzorcih vampnega soka. Za namnoževanje gena 16S rRNK vrsteP. bryantiismo uporabili univerzalni bakterijski začetni oligonulkleotid EUB 338 ter za to vrsto specifičen začetni oligonukleotid PBB14. Pripravili smo interno kontrolo, ki je imela isti prepoznavni mesti za začetna oligonukleotida na 5' in 3'. Od tarčne DNA, tj. genomske DNK vrsteP.bryantiiseva B14, se je razlikovala le po deleciji 41 bp na sredini nukleotidnega zaporedja. Namnožene produkte cPCR smo ovrednotili s kapilarno elektroforezo in rezultate izračunali z regresijsko enačbo (Reilly in Attwood, 1998). Na osnovi sočasnega pomnoževanja tarčne DNK, ki smo jo izolirali iz znanega števila celic čiste kultureP.bryantiiseva B14 in interne kontrole, smo izdelali umeritveno premico, ki omogoča številčno vrednotenje bakterijskih celicP.bryantiiv območju 2,15 x 103do 4,3 x 104.