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


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 10,000 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.


CONCLUSIONS
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.
van Beckhoven
J R C M
JR
Plant Research International, Wageningen, The Netherlands. J.R.C.M.vanBeckhoven@plant.wag-ur.nl
Stead
D E
DE
van der Wolf
J M
JM
D000192:Actinomycetales / Q000235:genetics* / Q000302:isolation & purification*; D001483:Base Sequence; D002470:Cell Survival; D003199:Computer Systems; D004269:DNA, Bacterial / Q000032:analysis* / Q000145:classification; D008969:Molecular Sequence Data; D021141:Nucleic Acid Amplification Techniques; D016133:Polymerase Chain Reaction / Q000379:methods; D012150:Polymorphism, Restriction Fragment Length; D012336:RNA, Ribosomal, 16S / Q000032:analysis* / Q000145:classification; D012680:Sensitivity and Specificity; D016415:Sequence Alignment; D011198:Solanum tuberosum / Q000382:microbiology*
D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S