DETECTION OF GENETICALLY-ENGINEERED MICROORGANISMS IN PADDY SOIL USING A SIMPLE AND RAPID NESTED POLYMERASE CHAIN-REACTION METHOD

A simple method for the detection of small populations of Pseudomonas fluorescens P.B8-1, containing the npt II gene of Tn5 as a unique marker, was applied to a Nyuzen paddy soil using cell extraction (indirect DNA extraction) and a ''nested'' polymerase chain reaction (PCR). This involved processing samples through a combination of a sucrose gradient centrifugation procedure to isolate bacterial cells, followed by cell lysis with proteinase K and CTAB (hexadecyltrimethyl ammonium bromide)-NaCl. This method allowed the extraction of DNA within about 6 h followed by amplification of DNA. The optimized ''nested'' PCR comprised a ''2-step'' PCR (45 cycles) using two 20-mer primers, followed by a ''3-step'' PCR (30 cycles) using two 26-mer primers which were internal to the first set. After the first PCR step was performed, the amplified DNA was detectable from the inoculated soil containing a minimum of 10(5) cfu g-1. However, the ''nested'' PCR procedure permitted the detection of amplified DNA fragments from inoculated non-sterile soils containing 1.3 x 10(1) cfu g-1. The application of this detection strategy was tested by monitoring the survival of P. fluorescens P.B8-1 in a non-sterile paddy soil during a 53-day period. The P.B8-1 population decreased in soils maintained at either 25 or 10-degrees-C after inoculation. After 53 days, samples of soil maintained at 10-degrees-C contained 10(2) cfu g-1 of P.B8-1 (as determined by selective plate count) and permitted amplification of DNA by the ''nested'' PCR. At the same time, P.B8-1 was not detected in soil maintained at 25-degrees-C by either method. The results obtained using this detection strategy suggest that it is highly applicable to monitoring the fate of genetically engineered microorganisms in natural paddy soils.