3.3.17
SENSITIVE DETECTION OF GRAPEVINE VIRUS A BY TIME-RELEASE PCR

R NAKAUNE and J IMADA

National Institute of Fruit Tree Science, Persimmon and Grape Research Center, Akitsu, Hiroshima 729-2494, Japan

Background and objectives
Reverse-transcription (RT)-PCR is a very useful tool for detection of plant viruses. However, it is difficult to amplify virus genomes from a sample containing virus at low concentration or high amounts of interfering plant substances. Recently, a time-release PCR protocol was developed to amplify low-copy-number DNA [1]. This study compares the sensitivity of time-release PCR and routine PCR in amplification of cDNA of grapevine virus A (GVA).

Materials and methods
Sample preparation: petioles from GVA-infected Vitis vinifera cv. Koshu were used for this experiment. Sample preparation was according to the method previously described [2]. The sample was then clarified with 1/3 volume of chloroform.
RT-time release PCR: DNA primers specific for GVA were constructed based on the nucleotide sequences corresponding to coat protein [3]. The sequences are 5'-CgATAACCACAACTgATCTgC-3' (375H) and 5'-gTACATATTACggTCgTAgCC-3' (375C), resulting in a 375-bp amplified fragment. To prepare viral RNA extracts for RT-PCR, 4 l of each serial dilution of clarified sample were diluted with sterile water and Triton X-100 to a volume of 10 l and a final Triton X-100 concentration of 1%. These samples were incubated at 99C for 5 min, then chilled on ice immediately. 1 l of extract was added to an RT-reaction mixture (RNA PCR Kit, TaKaRa) containing complementary primers to final volume of 10 l. The RT reaction was performed with the following parameters: annealing and extension at 50C for 15 min, and inactivation of enzyme at 99C for 5 min. 2 l of the cDNA mixture was added to 1 l of a PCR reaction mixture [1] containing 10 pmol of the homologous primer and 0.5 units of AmpliTaq Gold (Perkin-Elmer Cetus). The time-release PCR was performed with the following cycling parameters: pre-activation at 94C for 3 min, followed by 60 cycles of denaturation at 94C for 0.5 min, annealing at 62C for 0.5 min, and extension at 72C for 1 min, and a final extension at 72C for 7 min. The routine PCR was performed with the following cycling parameters: pre-activation at 94C for 12 min, followed by 40 cycles of denaturation at 94C for 0.5 min, annealing at 62C for 0.5 min, extension at 72C for 1 min, and a final extension at 72C for 7 min. The amplified DNA fragments were detected by 1.5% agarose-gel electrophoresis following by staining with ethidium bromide.

Results and conclusion
The new application using AmpliTaq Gold was used to detect GVA. The sensitivity of time-release PCR for detection of GVA was determined through a series of dilutions of infected grapevine tissue extract. This method allows the amplification of GVA genome after cDNA synthesis from 1/100 dilution. However, it was difficult to amplify a GVA genome by routine PCR methods from 1/10 dilution, probably because of the low concentration of virus. In conclusion, time-release PCR after cDNA synthesis may be a useful tool for the detection of virus genomes.

References
1. Kebelmann-Betzing C, Seeger K, Dragon S et al., 1998. BioTechniques 24, 154-158.
2. Minafra A, Hadidi A, 1994. Journal of Virological Methods 47, 175-188.
3. Minafra A, Saldarelli P, Grieco F, Martelli GP, 1994. Archives of Virology 137, 249-261.