Polymerase chain reaction and gene probe detection of the 2,4- dichlorophenoxyacetic acid degradation plasmid, pJP4

Julia W Neilson, K. L. Josephson, S. D. Pillai, Ian L Pepper

Research output: Contribution to journalArticle

31 Citations (Scopus)

Abstract

Specific and sensitive detection of indigenous and introduced degradative organisms is an essential prerequisite to their use in remediation of toxic waste and soil systems. Procedures were employed for the use of polymerase chain reaction and gene probes for sensitive detection of the 2,4- dichlorophenoxyacetic-acid-degrading bacterium, Alcaligenes eutrophus JMP134(pJP4). Two 20-mer oligonucleotide primers were identified for amplification of a 205-bp region of the tfdB gene of pJP4, and optimum conditions for amplification were determined. Both the polymerase chain reaction amplification process and hybridization with the 5'-end-labelled probe were found to be specific to organisms containing plasmid pJP4 or its derivative pRO103. Detection limits were determined for the template supplied either as bacterial cells or purified plasmid DNA. The detection was sensitive up to an initial inoculum of 3,000 CFU or 156 pg of total plasmid DNA. However, when the amplified product was transferred to a nylon membrane and hybridized with the 5'-end-labelled probe, the detection sensitivity increased to 300 CFU or 15.6 pg of plasmid DNA. This sensitive detection method is more specific than use of traditional indicator media (M. A. Loos, Can. J. Microbiol. 21:104-107, 1975). An oligonucleotide (20 bases) complementary to a sequence internal to the 205-bp region was synthesized and utilized as a probe to confirm the specificity of the detection.

Original languageEnglish (US)
Pages (from-to)1271-1275
Number of pages5
JournalApplied and Environmental Microbiology
Volume58
Issue number4
StatePublished - 1992

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2,4-Dichlorophenoxyacetic Acid
2,4 dichlorophenoxyacetic acid
plasmid
polymerase chain reaction
2,4-D
plasmids
Plasmids
probe
Polymerase Chain Reaction
degradation
gene
Genes
DNA
amplification
genes
Alcaligenes
DNA Primers
Poisons
Cupriavidus necator
Nylons

ASJC Scopus subject areas

  • Environmental Science(all)
  • Biotechnology
  • Microbiology

Cite this

Polymerase chain reaction and gene probe detection of the 2,4- dichlorophenoxyacetic acid degradation plasmid, pJP4. / Neilson, Julia W; Josephson, K. L.; Pillai, S. D.; Pepper, Ian L.

In: Applied and Environmental Microbiology, Vol. 58, No. 4, 1992, p. 1271-1275.

Research output: Contribution to journalArticle

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