A spot-PCR technique for the detection of phloem-limited grapevine viruses

被引:47
作者
LaNotte, P
Minafra, A
Saldarelli, P
机构
[1] UNIV BARI,DIPARTIMENTO PROTEZ PIANTE,I-70126 BARI,ITALY
[2] CTR STUDIO CNR VIRUS & VIROSI COLTURE MEDITERRANE,I-70126 BARI,ITALY
关键词
grapevine viruses; detection; diagnosis; spot-PCR;
D O I
10.1016/S0166-0934(97)00038-4
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
Specific amplification of genomic fragments of grapevine trichovirus A (GVA), grapevine trichovirus B (GVB) and grapevine leafroll-associated closterovirus 3 (GLRaV3) was obtained by reverse transcription-PCR on total nucleic acid solubilized from small pieces of charged nylon membrane, on which a drop of crude infected grapevine sap was spotted (spot-PCR). A thermal treatment (95 degrees for 10 degrees) of spotted sap in a buffered solution improved the release of viral template. Consistent amplification was obtained with three viruses from fragments of the same respective blots up to 1 month after spotting, while a detection threshold limit comparable with standard PCR techniques was found for this method. Duplex PCR (i.e. amplification of different viruses from a mixed-infected grapevine source) was also found to be effective, since GVA and GLRaV3 were amplified by a mixture of specific primers in the same reaction. This rapid and easy sampling technique, using leaf petioles to express crude sap, may have a wide field application for screening grapevine viruses. (C) 1997 Elsevier Science B.V.
引用
收藏
页码:103 / 108
页数:6
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