జర్నల్ ఆఫ్ ప్లాంట్ పాథాలజీ & మైక్రోబయాలజీ

నైరూప్య

A Simple, Rapid and Efficient Method of Pepino mosaic Virus RNA Isolation from Tomato Fruit

Amal Souiri, Mustapha Zemzami, Khadija Khataby, Hayat Laatiris, Saaid Amzazi and Moulay Mustapha Ennaji

The main concern in molecular detection of RNA viral pathogens in plants is the achievement of good quality of the extracted RNA. Various methods of isolating RNAs from both polysaccharide-rich and poor tissues and other recalcitrant plants are available. However, the use of time and reagent consuming methods and those involving hazardous chemicals is somewhat cumbersome and problematic, especially when it is not necessary for specific purposes like isolating viral RNA from tomato fruit, hence the objective of this paper. We describe an alternative, simple and rapid method for preparing viral RNA from tomato fruit without RNA extraction and purification steps, case of Pepino mosaic virus (PepMV).

The method employs mechanical treatment and suspension in water. The quality of RNA obtained was judged by spectrometric readings and validated in RT-PCR assays. The used protocol was compared with the usual TRIzol method. The results showed that the yield and the quality of RNA obtained using the proposed method are efficient and highly yielded in comparison with TRIzol method. Moreover, the developed method successfully allowed a sensitive and reproducible detection of PepMV predicted bands in RT-PCR. Thus, molecular detection of PepMV from tomato fruit can be performed routinely without fastidious RNA isolation. As well, this will make the diagnosis of other RNA viruses infecting tomato crops easier and less time-consuming, in comparison with the other methods performed with expensive commercial kits and those involving toxic chemicals. Finally, the described established method will contribute effectively in strategies of phytosanitary and certification programs of tomato crops worldwide.