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Related Experiment Videos

Frankia genus-specific characterization by polymerase chain reaction.

P Simonet1, M C Grosjean, A K Misra

  • 1Laboratoire de Microbiologie des Sols, U.R.A. Centre National de la Recherche Scientifique 1450, Université Lyon I, Villeurbanne, France.

Applied and Environmental Microbiology
|November 1, 1991
PubMed
Summary

This study developed a Polymerase Chain Reaction (PCR) test to identify Frankia bacteria in plant root nodules. The PCR assay accurately distinguishes Frankia from contaminants using specific gene targets.

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Area of Science:

  • Microbiology
  • Molecular Biology
  • Plant Science

Background:

  • Actinorhizal symbiosis is crucial for nitrogen fixation in plants.
  • Accurate identification of Frankia bacteria in root nodules is essential for ecological and agricultural studies.
  • Distinguishing Frankia from other microorganisms can be challenging using traditional methods.

Purpose of the Study:

  • To develop a rapid and sensitive Polymerase Chain Reaction (PCR) based assay for the specific identification of Frankia strains.
  • To differentiate Frankia from contaminating microorganisms within actinorhiza (nodules).
  • To validate the PCR assay's efficiency using isolates from Alnus nepalensis nodules.

Main Methods:

  • Utilized primer sets targeting specific DNA regions: nif genes (nifH, nifD) and rRNA genes (16S, 23S).

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  • Employed degenerate oligonucleotide primers for broad amplification of nitrogen-fixing microorganisms and specific primers for Frankia.
  • Incorporated universal and specific primers for rRNA gene regions, tested against diverse microbial DNAs.
  • Performed agarose gel electrophoresis to detect amplified DNA fragments and confirm specificity.

    • Main Results:

    • Successfully developed a PCR assay capable of differentially amplifying DNA from Frankia strains.
    • Demonstrated high specificity for Frankia using primers targeting nif and rRNA gene regions.
    • Validated the assay with an isolate from Alnus nepalensis, confirming its accuracy against nodulation tests.
    • Established effective positive and negative controls for reliable discriminant amplification.

    Conclusions:

    • The developed PCR assay provides a rapid, sensitive, and specific method for identifying Frankia bacteria in plant nodules.
    • This molecular tool can reliably distinguish Frankia from contaminating microorganisms, aiding in symbiotic studies.
    • The assay's efficiency is confirmed, offering a valuable alternative to traditional methods for Frankia identification.