Detection of genetically engineered microorganisms in paddy soil using a simple and rapid "nested" polymerase chain reaction method

Tsushima, Seiya and Hasebe, Akira and Komoto, Yukiomi and Carter, John P. and Miyashita, Kiyotaka and Yokoyama, Kazunari and Pickup, R. W. (1995) Detection of genetically engineered microorganisms in paddy soil using a simple and rapid "nested" polymerase chain reaction method. Soil Biology and Biochemistry, 27 (2). pp. 219-227. ISSN 0038-0717

Abstract

A simple method for the detection of small populations of Pseudomonas fluorescens P.B8-1, containing the nptII 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 105 cfu g-1. However, the "nested" PCR procedure permitted the detection of amplified DNA fragments from inoculated non-sterile soils containing 1.3 × 101 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°C after inoculation. After 53 days, samples of soil maintained at 10°C contained 102 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°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.