Rapid Detection of Grapevine Leafroll-associated virus Type 3 using the reverse transcription loop-mediated amplification method

[摘要] Grapevine Leafroll disease (GLD), one of the most destructive diseases ofgrapevines, has been found in every country where grapevines are grown.Grapevine Leafroll associated virus type 3 (GLRaV-3), one of several virusesassociated with GLD globally, is the most prevalent virus in South African grapevinesand therefore control of GLRaV-3 takes high priority in any strategy aimed at controlof GLD. GLD can be controlled through the use of an integrated strategy whichincludes using certified plant material, controlling insect vectors through use ofsystemic insecticides and the removal of infected vines by roguing. Infectedindividuals are identified each autumn, using either symptom display (in red cultivars,where infected individuals display interveinal reddening and downward rolling ofleaves) or ELISA (in symptomless white cultivars). ELISA is laborious, timeconsuming and relatively insensitivity compared to molecular techniques and asimpler, more rapid and more sensitive means of indentifying GLRaV-3 infectedvines is required.A simple RNA extraction procedure combined with a single-tube reversetranscriptase loop-mediated amplification (RT-LAMP) has been developed whichallows for the rapid, simple detection of GLRaV-3. Using RT-LAMP, a viral target canbe amplified in 2 hours under isothermal conditions. This GLRaV-3 specific RTLAMPuses hydroxy napthol blue (HNB), a colourimetric indicator that changes fromviolet to sky blue only where a positive RT-LAMP reaction has occurred, makingresults quick and easy to interpret. The sensitivity of this technique was compared toELISA and nested PCR by pooling samples at varying ratios of healthy to infectedplants. Using nested PCR and RT-LAMP 1 infected sample could be detectedamongst 50 healthy individuals while ELISA could only detect 1 amongst 30 infectedmaking RT-LAMP more sensitive than ELISA. Further RT-LAMP could be performedin 2 hours compared to nested PCR and ELISA's 8 and 48 hours respectively. Basedon these results, RT-LAMP is viable alternative for ELISA for the detection ofGLRaV-3 in the field.RT-LAMP was also tested for its ability to detect GLRaV-3 in grapevine rootstockswhere, due to low viral titres and erratic distribution, it is notoriously difficult to detect. The rootstocks which were used for testing of GLRaV-3 had been tested in aprevious study and it was found that only 28% of samples tested positive after 33months (post inoculation). Using RT-LAMP, 78% of samples tested positive forGLRaV-3. Although further testing must be done, RT-LAMP may also be a viablealternative for testing grapevine rootstocks for GLRaV-3 infection.

[发布机构] University of Pretoria