Detection of Clavibacter michiganensis subsp. sepedonicus by AmpliDet RNA, a new technology based on real time monitoring of NASBA amplicons with a molecular beacon

• Published in: Journal of applied microbiology Vol. 93; no. 5; pp. 840 - 849
• Main Authors: Beckhoven, J.R.C.M. van, Stead, D.E, Wolf, J.M. van der
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Science Ltd 01-01-2002; Blackwell Science; Oxford University Press
• Subjects: Actinomycetales - genetics; Actinomycetales - isolation & purification; bacterial ring rot; Bacteriological methods and techniques used in bacteriology; Bacteriology; Base Sequence; Biological and medical sciences; Cell Survival; cell viability; cell walls; Clavibacter michiganensis subsp. sepedonicus; Computer Systems; detection limit; DNA; DNA, Bacterial - analysis; DNA, Bacterial - classification; epidemiology; Fundamental and applied biological sciences. Psychology; Microbiology; Molecular Sequence Data; monitoring; Nucleic Acid Amplification Techniques; nucleotide sequences; phenol; Plant Research International; Polymerase Chain Reaction - methods; Polymorphism, Restriction Fragment Length; ribosomal RNA; risk; RNA, Ribosomal, 16S - analysis; RNA, Ribosomal, 16S - classification; Sensitivity and Specificity; Sequence Alignment; Solanum tuberosum - microbiology; technology; Bacteria; Actinomycetes; Method; Detection; Clavibacter michiganensis
• DOI: 10.1046/j.1365-2672.2002.01765.x
• EISSN: 1365-2672
• ISSN: 1364-5072

Aims: To develop a procedure for direct detection of viable cells of Clavibacter michiganensis subsp. sepedonicus (Cms), the causal organism of bacterial ring rot in potato, based on AmpliDet RNA, in which amplicons generated by nucleic acid sequence based amplification (NASBA) are monitored in real time with a molecular beacon. Methods and Results: Five methods were evaluated and fine-tuned for extraction of RNA from Cms. The most efficient non-commercial RNA extraction method included an enzymatic breakdown of the cell wall followed by a phenol extraction. AmpliDet RNA enabled detection of 10000 molecules of purified rRNA per reaction and 100 cfu of Cms per reaction in more complex samples. Two primer pairs were tested with DNA and RNA purified from Cms. One primer pair was able to distinguish live from dead cells. Conclusions: An AmpliDet RNA was developed which enabled fast and specific detection of viable cells of Cms in complex substrates at a detection limit of 100 cfu per reaction. Significance and Impact of the Study: This novel AmpliDet RNA is carried out in sealed tubes, thus reducing the risk of carry-over contamination. The method will be particularly suitable for studies on the epidemiology of Cms in which viable cells should be exclusively detected.