Fluorescence resonance energy transfer (FRET) based molecular detection of a genetically modified PCB degrader in soil.
Genetic analysis of the location of a mini-Tn5 promoted insertion of the LB400 bph operon in the rhizosphere coloniser Pseudomonas fluorescens F113rifPCB, allowed the development of a specific PCR detection system based on the unique DNA sequence at this insertion site. Real time PCR using both SYBR green chemistry and Fluorescence Resonance Energy Transfer probes allowed the precise identification of the recombinant strain and its quantitative detection in soil microcosms over a (bacteria/g) range of five orders of magnitude. This new assay can detect the genetically modified microorganism from soil in less than 90 min and at levels below the detection limits of standard PCR or cultivable counts on selective media.
Hogan
Jill
J
Department of Applied Biology and Chemistry, Institute of Technology Carlow, Kilkenny Road, Carlow, Ireland.
Sherlock
Orla
O
Ryan
David
D
Whelan
Clare
C
Francesconi
Stephen
S
Rivilla
Rafael
R
Dowling
David N
DN
D052160:Benzothiazoles; D001673:Biodegradation, Environmental; D002874:Chromosome Mapping; D004251:DNA Transposable Elements / Q000235:genetics; D004269:DNA, Bacterial / Q000737:chemistry / Q000302:isolation & purification; D003959:Diamines; D005453:Fluorescence; D031541:Fluorescence Resonance Energy Transfer; D023061:Gene Order / Q000235:genetics; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D008969:Molecular Sequence Data; D009876:Operon / Q000235:genetics / Q000502:physiology; D009930:Organic Chemicals / Q000032:analysis; D011078:Polychlorinated Biphenyls / Q000378:metabolism*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011551:Pseudomonas fluorescens / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D011804:Quinolines; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D012988:Soil Microbiology
D052160:Benzothiazoles; D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D003959:Diamines; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I; D011078:Polychlorinated Biphenyls