A real-time RT-PCR assay for the detection and quantification of grapevine leafroll-associated virus 3 (GLRaV-3) in Vitis vinifera (Vitaceae) and Planococcus ficus (Signoret) (Hemiptera : Pseudococcidae)

Abstract

Viral diseases represent a major obstacle to commercial growing of grapevines. Grapevine leafroll-associated virus 3 (GLRaV-3) is one of the most economically important viral diseases in South African vineyards. The most important vector of GLRaV-3 in South Africa is the vine mealybug Planococcus ficus. Studies show that single first instar-nymphs can successfully transmit GLRaV-3 to healthy grapevines. Quantifying the amount of virus acquired in relation to feeding time of mealybugs may lead to a better understanding of GLRaV-3 mealybug transmission. In this study, a real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay was developed to quantify GLRaV-3 in grapevines and mealybugs. Real-time qRT-PCR is a highly sensitive technique. The quality of purified RNA and the accuracy of the quantification technique are essential for reliable quantitative use. To develop a real-time qRT-PCR assay two tasks were undertaken. Firstly, four different RNA isolation techniques, namely phenolchloroform, Gentra Purescript® RNA Isolation kit, Qiagen QIAzol™ and Qiagen RNeasy® Plant Mini kit, were assessed for use in qRT-PCR. Phenol-chloroform RNA extraction method was superior to the other RNA extraction methods tested, with regard to purity of RNA, RT-PCR efficiency, reproducibility and number of GLRaV-3 positive samples detected. Secondly, DNA and cRNA external standard models were designed for quantifying GLRaV-3 in grapevines and mealybugs. External standards have defined concentrations of a target nucleic acid and are used to generate a standard curve. The DNA standard model had a wider detection and quantification range than the cRNA standard model. However, the DNA standard model, unlike the cRNA standard model is not subjected to the RT step. Therefore, using the DNA standard model, GLRaV-3 is quantified based on the starting cDNA concentration and not the initial RNA concentration. The cRNA standard model provides information on the initial RNA concentration. The DNA standard model is best applied as a quantification method where initial RNA concentration is not relevant. The cRNA standard model can be applied for accurate quantification of GLRaV-3 in grapevines and mealybugs. The real-time qRT-PCR assay designed in this study can be applied in other laboratories. Copyright