Review Article Souiri et al., J Plant Pathol Microbiol 2017, 8:2 DOI: 10.4172/2157-7471.1000395 Journal of Plant Pathology & Microbiology J o u r n a l o f P l a n t P a t h ol o g y & M i c r o b i o l o g y ISSN: 2157-7471 Research Article Open Access Volume 8 • Issue 2 • 1000395 J Plant Pathol Microbiol, an open access journal ISSN: 2157-7471 Keywords: Tomato; RNA isolation; Pepino mosaic virus; Molecular diagnosis Introduction Tomato (Solanum lycopersicum) is one of the most widely grown vegetable crops. In many worldwide regions where tomato is cultivated, viral diseases have become one of the main limiting factors in tomato production. Over the past few years, Pepino mosaic virus (PepMV), genus Potexvirus, Family Flexiviridae, has caused signifcant economic losses in tomato production areas in Europe [1-4], North America [5], South America [6], and Asia [7]. Detection and prevention are the main control measures. Diagnosis based on disease symptoms is not reliable, because symptoms can vary according to the PepMV isolates [8]. Several methods have been developed for the detection of PepMV in plants. Serological techniques like ELISA have been developed and used successfully for a number of years for the detection of plant viruses. Recently, the trend toward molecular biology techniques such as RT-PCR and nucleotide sequencing has risen in last years. Te use of RT-PCR in diagnosing PepMV is described in Hasiów et al. [9]. An immunocapture-retrotranscription-PCR (IC-RT-PCR) approach is detailed in Mansilla et al. [10]. Prior to RT-PCR, RNA virus isolation is a critical step. For the majority of plant material containing high levels of polysaccharides and polyphenols, pigments and RNase is a challenging starting material for high-quality RNA isolation in reasonable amounts because of diferent amounts of those substances in diverse tissues. A large number of methods have been developed or widely modifed [11,12] for RNA isolation and purifcation from plant tissues and adopted by researchers and laboratories. Te RNA isolation method developed by Chomcynski and Sacchi [11] employ a single extraction with acid guanidinium thiocyanate-phenol-chloroform mixture [11]. Tis method incorporated TRIzol, a ready-to-use reagent. Since its introduction, this method has become widely used for isolating total RNA from biological samples of diferent sources, during last few decades [13]. Tompson et al. [14] used the Plant RNeasy Kit produced by QIAGEN to quickly extract high-quality total RNA from strawberry leaves, but the kit is expensive [14]. In addition, protocols designed for plant are time consuming, require hazardous products and are tissue specifc [12,15,16]. In the case of tomato, the level of polysaccharides and other interfering components varies in dependence of fruit ripening stages and nature of tissue (leaf, stem, and root, fower, fruit). Consequently, the yield of total RNA extracted difers from a tomato tissue to another [15]. Te method described by Wang et al. [15] for isolation of total RNA from tomato fruit was used in gene expression studies at the microarray-based level [15]. However, isolation of viral RNA from infected tomato fruit is not as difcult and requiring as isolation of functional RNA and small RNAs [17]. Optimizing protocols of RNA virus isolation step, is important in order to meet sensitivity, specifcity and rapid means of detecting RNA plant viruses, including PepMV, in tomato tissues. A Simple, Rapid and Efficient Method of Pepino mosaic Virus RNA Isolation from Tomato Fruit Amal Souiri 1-3 , Mustapha Zemzami 3 , Khadija Khataby 1 , Hayat Laatiris 3 , Saaid Amzazi 2 and Moulay Mustapha Ennaji 1 * 1 Laboratory of Virology, Microbiology, Quality, and Biotechnologies/Ecotoxicology and Biodiversity, Faculty of Science, and Techniques Mohammedia, University Hassan II of Casablanca, Morocco 2 Laboratory of Biochemistry and Immunology, Faculty of Sciences, University of Mohammed V, Rabat, Morocco 3 Laboratory of Sanitary Control, Control Unit of Plants, Domaines Agricoles Maâmora, Salé, Morocco Abstract 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 specifc 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 purifcation 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 effcient 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 certifcation programs of tomato crops worldwide. *Corresponding author: Moulay Mustapha Ennaji, M.Sc. Ph. D RSM (CCM) Laboratory of Virology Microbiology, Quality and Biotechnologies /Ecotoxicology and Biodiversity, Faculty of Science and Techniques Mohammedia, University Hassan II of Casablanca, Mohammedia 20650, Morocco, Tel: +212661748862/ +212662013772; E-mail: m.ennaji@yahoo.fr Received January 16, 2017; Accepted January 30, 2017; Published February 21, 2017 Citation: Souiri A, Zemzami M, Khataby K, Laatiris H, Amzazi S, et al. (2017) A Simple, Rapid and Effcient Method of Pepino mosaic Virus RNA Isolation from Tomato Fruit. J Plant Pathol Microbiol 8: 395. doi: 10.4172/2157-7471.1000395 Copyright: © 2017 Souiri A, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.