Real time PCR as a versatile tool for virus detection and transgenic plant analysis

[摘要] ENGLISH ABSTRACT: South Africa is regarded as one of the top wine producing countries in the world.One of the threats to the sustainability of the wine industry is viral diseases of whichGrapevine leafroll-associated virus 3 (GLRaV-3) and Grapevine virus A (GVA) areconsidered to be the most important and wide spread. Scion material is regularlytested for viruses; however scion material is often grafted onto rootstocks that havequestionable phytosanitary status. Virus detection in rootstocks is challenging due tolow and varying titres, but is imperative as a viral control mechanism. An additionalviral control mechanism is the use of transgenic grapevine material which offersresistance to grapevine infection.The objective of this project was to establish a detection system using real time PCR(qPCR) techniques, to accurately and routinely detect GLRaV-3 and GVA inrootstock propagation material. qPCR would furthermore be used to performmolecular characterisation of transgenic plants containing a GLRaV-3 antiviralΔHSP-Mut construct.A severely infected vineyard (Nietvoorbij farm) in the Stellenbosch area wasscreened throughout the grapevine growing season to investigate virus prevalencethroughout the season and to determine the optimal time for sensitive virus detection.A large scale screening of nursery propagation material for GLRaV-3 infection wasalso conducted. The qRT-PCR results were compared to DAS-ELISA results tocompare the efficacy and sensitivity of the two techniques. For the severely infectedvineyard, the ability to detect GLRaV-3 increased as the season progressed towardswinter. qRT-PCR was more sensitive and accurate in detecting GLRaV-3 than DASELISA,as the latter technique delivered numerous false positive results later in theseason. The best time to screen for GLRaV-3 in the Western Cape region was fromthe end of July to September. For the nursery screenings, our qRT-PCR results werecompared to the results of the DAS-ELISA performed by the specific nurseries. NoGLRaV-3 infection was detected in the specific samples received from the twodifferent nurseries. The results for all the samples correlated between the two techniques. This confirms that the propagation material of these nurseries has ahealthy phytosanitary status with regards to GLRaV-3.However, the detection of GVA in the severely infected vineyard yielded inconsistentresults. Detection ability fluctuated throughout the season and no specific trend inseasonal variation and virus titre fluctuation could be established. The highestpercentage of GVA infected samples were detected during September, April and theend of July. Previously published universal primers were used for the detection ofGVA, but further investigation indicated that they might not be suitable for sensitivedetection of specific GVA variants present in South Africa.Vitis vinifera was transformed with a GLRaV-3 antiviral construct, ΔHSP-Mut.SYBR Green Real time PCR (qPCR) and qRT-PCR were utilised as alternativemethods for molecular characterisation of transgenic plants. The qPCR and Southernblot results correlated for 76.5% of the samples. This illustrated the ability of qPCRto accurately estimate transgene copy numbers. Various samples were identifiedduring qRT-PCR amplification that exhibited high mRNA expression levels of thetransgene. These samples are ideal for further viral resistance studies.This study illustrated that the versatility of real time PCR renders it a valuable tool foraccurate virus detection as well as copy number determination.

[发布机构] Stellenbosch University