Articles
DETECTION OF CUCUMBER MOSAIC AND TOMATO ASPERMY CUCUMOVIRUSES BY MEANS OF POLYMERASE CHAIN REACTION
However, the utilization of hybridization techniques in plant pathogen diagnosis has been limited so far, indicating that several improvements of the current procedures are required for routine applications.
Recently, an enzymatic procedure named polymerase chain reaction (PCR) has been described which allows the amplification of very low amounts of target nucleic acids and, therefore, has been used successfully to detect minute amounts of viral nucleic acids including plant viruses.
Our first attempts to adapt the PCR technology to the detection of viruses in ornamental plants were directed to the diagnosis of cucumoviruses, because their particles are known to be quite unstable in crude extracts.
We expected the experience gathered on this system could then be translated to other viruses.
Virus isolates were obtained from F. Rabenstein (Aschersleben) and G. Adam (Braunschweig). The chosen primer sequences belonged to the intergenic region of RNA 3 (5′-primer) and the 3′- proximal part of the coat protein gene (3′-primer) of both viruses.
They correspond to higly conserved regions.
Total RNA was isolated from infected and healthy plant material following a DIAGEN protocol using QIAGEN minicolumns.
The first strand cDNA was synthesized at 37°C in a 20 ul reaction mix containing 0,25 umol 3′-primer, 0.25 mM of each dNTP, 20 U RNasin (Boehringer), 200 U M-MLV-reverse transcriptase (BRL) and the corresponding RT-reaction buffer (BRL). After 15 min the amplification mix was added.
This mix contained, in a final volume of 100 ul, 0.25 umol of each primer, 0.25 mM of each dNTP, 2.5 U Taq-DNA polymerase (Boehringer) and the standard reaction buffer (Boehringer). Samples were overlaid with 70 ul kerosine and placed in a Perkin Elmer Cetus thermal cycler programmed to give 35 cycles at 94 °C (1 min) 52 °C (1,5 min) and 72 °C (1,5 min). Samples taken after amplification revealed specific bands of the expected size.
These DNA fragments were isolated from agarose gels following the QIAEX protocol.
Thex were subsequently cloned into the bluescript KS vector (Stratagene) in order to obtain the necessery standard RNA