+ Site Statistics
+ Search Articles
+ PDF Full Text Service
How our service works
Request PDF Full Text
+ Follow Us
Follow on Facebook
Follow on Twitter
Follow on LinkedIn
+ Subscribe to Site Feeds
Most Shared
PDF Full Text
+ Translate
+ Recently Requested

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



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



Soil Biology & Biochemistry 27(2): 219-227



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 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 times 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 degree C after inoculation. After 53 days, samples of soil maintained at 10 degree 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 degree 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.

Please choose payment method:






(PDF emailed within 0-6 h: $19.90)

Accession: 002591458

Download citation: RISBibTeXText

DOI: 10.1016/0038-0717(94)00162-t


Related references

Rapid method for the detection of genetically engineered microorganisms by polymerase chain reaction from soil and sediments. Journal of industrial microbiology and biotechnology 20(2): 90-94, 1998

A simple and rapid method for detection of Trypanosoma evansi in the dromedary camel using a nested polymerase chain reaction. Kinetoplastid Biology and Disease 5: 2, 2006

Detection of genetically modified soybeans in miso by polymerase chain reaction and nested polymerase chain reaction. Journal of Food and Drug Analysis 11(2): 154-158, 2003

Rapid and sensitive method for the detection of B19 virus DNA using the polymerase chain reaction with nested primers. Journal of Virological Methods 44(2-3): 221-234, 1993

A simple method for increase of the specificity of nested polymerase chain reaction for the detection of envelope sequence. Chinese Journal of Microbiology & Immunology 13(1): 63-64, 1993

Rapid and sensitive detection of equine arteritis virus in semen and tissue samples by reverse transcription-polymerase chain reaction, dot blot hybridisation and nested polymerase chain reaction. Acta Virologica 42(5): 333-339, 1998

A simple method for the detection of CYP2C9 polymorphisms: Nested allele-specific multiplex polymerase chain reaction. Clinica Chimica Acta 336(1-2): 97-102, 2003

Development and application of a nested polymerase chain reaction method for the detection of genetically modified soybean in Chinese traditional fermented soy food-sufu. Journal of Food and Drug Analysis 12(3): 266-272, 2004

A novel nested reverse-transcriptase polymerase chain reaction method for rapid hepatitis C virus detection and genotyping. Indian Journal of Medical Microbiology 32(2): 130-136, 2014

Enhanced detection of genetically engineered Corynebacterium glutamicum pUN1 in directly extracted DNA from soil, using the T4 gene 32 protein in the polymerase chain reaction. European Journal of Soil Biology 30(2): 93-98, 1994

Application of one tube reverse transcription and nested polymerase chain reaction (RT-nested PCR) for rapid detection and differentiation of pestiviruses. Veterinarija ir Zootechnika (18): 67-73, 2002

Enhanced detection of Prune dwarf virus in peach leaves by immunocapture-reverse transcription-polymerase chain reaction with nested polymerase chain reaction (IC-RT-PCR nested PCR). Journal of Phytopathology 150(2): 94-96, 2002

Increased sensitivity for detecting genetically engineered microorganisms in environmental samples by polymerase chain reaction. Abstracts of the Annual Meeting of the American Society for Microbiology 88: 284, 1988

The use of nested polymerase chain reaction method for rapid detection of cytomegalovirus in peripheral blood leukocytes of patients after bone marrow transplantation. Voprosy Virusologii 41(4): 147-149, 1996

Detection of genetically engineered Roundup ReadyTM soyabeans using the polymerase chain reaction (PCR). Preliminary examination of the method within the scope of the Subcommission 29a of the Swiss Food Manual. Mitteilungen aus dem Gebiete der Lebensmitteluntersuchung und Hygiene 88(6): 722-731, 1997