Sensitive, fast and easy detection of Grapevine fleck virus from crude extracts by reverse transcription loop-mediated isothermal amplification (RT-LAMP)

Grapevine fleck virus (GFkV) is the causing agent of fleck disease and occurs in grapevine worldwide. Infection is latent in most scion and rootstock cultivars but can lead to several symptoms such as translucent spots and clearing of veins in leaves. Symptoms typically increase when infections with multiple virus species occur. Excluding infected grapevines from propagation is the only possibility to control the spread of this disease. Consequently, sensitive, inexpensive and fast methods for the detection of this virus are needed. In the present study, a detection method for GFkV was developed employing reverse transcription loop-mediated isothermal amplification (RT-LAMP), a method to reverse transcribe viral RNA and amplify DNA in one step. Primers were designed to bind the coat protein gene. The RT-LAMP works without the need for time consuming RNA extraction. In contrast, a crude homogenate can be used directly to set up the RT-LAMP reaction. Sensitivity of RT-LAMP was compared to ELISA by diluting extracts from infected material in extracts prepared from healthy plants. While ELISA gave positive results for all undiluted samples and most samples diluted 1:10, RT-LAMP was positive for all samples diluted up to 1:100 and gave a positive signal even after diluting 1:1.000.000 in most cases. Thus, it is possible to identify also those plants with lower virus titers, which then can be excluded from further propagation.