ABSTRACT
Two inhibitors of Taq DNA polymerase were isolated from the marine red alga Symphyocladia latiuscula. The inhibitors were purified by methanol extraction, molecular fractionation below 3000 MW and reverse-phase HPLC. The purified compound SL-1 containing three bromines was identified as 2,3,6-tribromo-4,5-dihydroxybenzyl alcohol (C7H5Br3O3: MW374) by NMR and MS analyses. The purified compound SL-2 was identified as 2,3, 6-tribromo-4,5-dihydroxybenzyl methyl ether(C8H7Br3O3: MW388). In a 25-microl reaction mixture containing 1.5 units of Taq DNA polymerase, the enzyme was completely inhibited by 0.5 microg SL-1 or 5 microg SL-2.
Subject(s)
Rhodophyta/chemistry , Base Sequence , Chromatography, High Pressure Liquid , Electrophoresis, Agar Gel , Enzyme Inhibitors/chemistry , Ethers , Hydrocarbons, Brominated/chemistry , Methanol/chemistry , Molecular Weight , Polymerase Chain Reaction , Spectrum Analysis , Taq Polymerase/antagonists & inhibitorsABSTRACT
A commercial kit intended for Taq polymerase inhibitor removal was tested to detect Shiga-toxigenic Escherichia coli (STEC) by polymersase chain reaction (PCR) directly from cattle fecal samples. Forty-five samples were analysed for the presence of stx genes. Results were compared to those obtained by two other methods: amplification of DNA purified by a non-commercial procedure (heat lysis protocol), and amplification of DNA from samples cultured in solid media, commonly used in our lab. Identical numbers of positive samples (33/45, 73 %) were obtained with the QIAamp DNA stool purification kit and the culturing procedure, suggesting an adequate removal of inhibitors that interfere in PCR amplification from the feces. Besides, the number of positive samples detected using DNA purified by the non-commercial protocol was lower, 25/39 (64%) than that achieved by using the kit. In conclusion, the use of the QIAamp DNA stool purification kit provided a rapid stx gene detection by PCR in bovine fecal samples.
Un kit comercial diseñado para la eliminación de inhibidores de la polimerasa Taq fue ensayado para la detección de STEC por PCR en muestras fecales de bovinos. Cuarenta y cinco muestras fueron evaluadas por la presencia de genes stx. Los resultados fueron comparados con aquéllos obtenidos por otros dos métodos: amplificación de ADN purificado por un procedimiento no comercial (protocolo de lisis por calor), y amplificación de ADN de muestras cultivadas en medio sólido, comúnmente usado en nuestro laboratorio. El mismo número de muestras positivas (33/45, 73 %), fueron obtenidas con el QIAamp DNA stool purification kit y el procedimiento de cultivo, sugiriendo una eliminación adecuada de inhibidores que interfieren con la amplificación en materia fecal. Por otro lado, el número de muestras positivas detectadas usando ADN purificado por el protocolo no comercial fue menor, 25/39 (64%). En conclusión, el uso del kit QIAamp DNA stool purification permitió una detección rápida de genes stx por PCR en muestras fecales bovinas.