Attenuation of PCR inhibition in the presence of plant compounds by addition of BLOTTO.

Abstract

Amplification of DNA extracted from complex milieus such as soil and plants by the polymerase chain reaction (PCR) is often limited by the presence of compounds inhibitory to the reaction (4). Inhibition is attributed to humic substances in soil which adsorb to various organic substances and form complexes with metal ions (1). Simple dilution of sample attenuates inhibition but also decreases test sensitivity when DNA template is a limiting factor (5). We encountered inhibition of PCR in experiments to detect low levels of plant pathogenic bacteria and fungi in plant tissue. The inhibition was probably due to plant polyphenolic molecules in the extracts. We found that the addition of non-fat milk eliminated the inhibitory effect in all of our samples. Non-fat milk cocktails, termed BLOTTO (Bovine Lacto Transfer Technique Optimizer, 3), have been widely used to prevent non-specific binding of proteins and nucleic acids to nitrocellulose in Western and Southern blotting procedures. BLOTTO is also used to inhibit non-specific binding in enzymelinked immunosorbent assays (2). In our experiments we extracted DNA from potato tuber tissue to detect Erwinia carotovora subsp. atmseptica and Clavibacter michiganensis subsp. sepedonicus the causal agents of the blackleg and ring rot diseases of potato, and cucumber roots for the presence of the damping-off pathogen, Pythium spp. Portions of potato tissue (0.5—2.0 g) were crushed and shaken with 1 ml of sterile distilled water in zip-lock plastic bags. One ml of fluid was removed and centrifuged in an Eppendorf tube at 17 300 g for 20 min and the pellet resuspended in 100 |il of Tris—EDTA buffer (pH 8.0) containing 1 % (w/v) SDS. The EDTA concentration was increased to 50 mM and the samples treated with Proteinase K at 10 ng/ml at 50°C for 3 h. Then one half volume of 7.5 M ammonium acetate was used to precipitate protein debris in the sample. Subsequently, DNA was precipitated from the supernatant fraction with I vol of isopropanol and then washed with 70% ethanol. Cucumber root samples were processed in a similar manner but after Proteinase K digestion they were treated with RNase and extracted with phenol/chloroform rather than being clarified by ammonium acetate treatment In each case the DNA fraction was dried at 58°C for 10 min, resuspended in 50 |il purified water and heated to 50-55°C prior to PCR.