Detection and enumeration of a genetically modified fungus in soil environments by quantitative competitive polymerase chain reaction
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abstract
A procedure of quantitative competitive polymerase chain reaction was developed for detection and quantitation of a genetically modified strain of Trichoderma virens (GvT6) in the soil environment. A 1.0-kb coding region of the opd (organophosphate degrading) gene on GvT6 genomic DNA was co- amplified with a standard DNA. The standard DNA was a 1.5-kb fragment of the gpd (glyceraldehyde-3-phosphate dehydrogenase) gene of T. virens containing opd-primer-binding sites on the ends. Soil samples containing various types of propagules of GvT6 were treated with a combined procedure of SDS-heating and bead-beating and the released nucleic acids were extracted and purified. Relative efficiency of T. virens DNA recovery from soil samples was about 85- 95% compared with DNA yield from spore suspensions. Following the PCR, the product ratio between the target and the standard was plotted against a dilution series of the standard which was added into the amplification reaction. Then, the concentration of the opd gene and the GvT6 in the soil sample were estimated. Despite the differences in size and nucleotide sequences between the target and the standard, they were amplified with similar efficiency. The detection limit of the PCR was 10-1000 times lower compared to traditional dilution plating. The quantitative competitive PCR technique will enhance our ability to understand the ecology of soilborne fungi in the soil environment.
