Monitoring system for the biocontrol agent Pichia anomala strain K using quantitative competitive PCR-ELOSA

Abstract

A quantitative-competitive PCR (QC-PCR) method with enzyme-linked oligosorbent assay (ELOSA) was developed to monitor Pichia anomala strain K, a biocontrol agent against postharvest diseases on apples. The procedure involved: (i) extraction of strain K DNA; (ii) coamplification of a constant amount of the extracted DNA (containing a strain K DNA marker of 262 base pairs (bp) specifically amplified with SCAR primers K1 and K2) with an internal standard (IS) titration; and (iii) differential hybridization with two specific biotinylated probes either for the target or for the IS sequence on microplate. The IS sequence differed from the target by only a short internal region of 35 bp providing the differential detection between both sequences. Both target and IS sequences proved to be competitive in PCR as well as in sandwich hybridization. Two copies of the target sequence were detected in the strain K genome by means of enzymatic restriction and Southern blot analysis. Varying amounts of strain K cell suspension were quantified in the phosphate buffer used for recovering cells from the apple surface. An accurate estimate of the strain K population was obtained from 103 to 106 yeast cells per apple. The threshold of the method was found to be at 1000 colony-forming units per apple, which was around 100 times more sensitive than the plating method for monitoring strain K.