Reverse transcription loop-mediated isothermal amplification (RT-LAMP) of plum pox potyvirus Turkey (PPV-T) strain

Abstract

Plum pox potyvirus (PPV) is one of the most important viral pathogens of stone fruit trees. PPV-T (Turkey) is important, unique and dominated strain in Turkish PPV pool. Due to the virus in internal quarantine pathogen in Turkey, the rapid detection of virus is too important for preventing the spread of the disease to new areas. In this study, a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay from Varga and James was adapted for the rapid detection of PPV-T isolates in Turkey. RT-LAMP assay did not show cross-reaction with other viruses commonly infects Prunus spp. or natural hosts of PPV-T. Assay could be completed with the help of a water bath under isothermal conditions (60 °C) in 35 min. Sensitivity analysis showed that RT-LAMP detected viral RNA concentrations up to 0.01 ng/µL and it was 10 times more sensitive than reverse transcription-polymerase chain reaction (RT-PCR). The optimal Mg+2 and dNTP concentration for RT-LAMP of PPV-T were 6 mM and 0.4 mM, respectively. Furthermore, assessment with the naked eye after staining of in-tube RT-LAMP products with SYBR Green I facilitated the interpretation of positive LAMP reactions. These results reveal that the method is more sensitive, faster, cheaper and gene-specific test for the detection of PPV-T than RT-PCR. This adaptation study may help in preventing epidemic spread of this destructive strain of virus in Turkey. This is the first study for the use of RT-LAMP application of the assay for plant viruses in Turkey.