3/14/2023 0 Comments Geneious prime 10The real-time RT-PCR protocol was carried out in a StepOnePlus thermal cycler (Applied Biosystems, Foster City, CA, USA). The amplification conditions consisted of an initial reverse transcription step at 45 ☌ for 45 min followed by a denaturation step at 95 ☌ for 10 min and 40 cycles of amplification (30 s at 95 ☌, 30 s at 50 ☌ and 25 s at 60 ☌) with a final elongation step at 60 ☌ for 7 min. The reaction was performed in a total volume of 25 μL using the master mix AgPath-ID™ (Ambion Inc., Austin, TX, USA) containing 500 nM of each primer and 50 ng of total purified RNA. The conventional RT-PCR was performed in the Veriti 96 Well thermal cycler (Applied Biosystems, Foster City, CA, USA). The 65 GLRaV-1-positive samples were tested by using two previously described protocols, a conventional RT-PCR and one real-time RT-PCR. In Europe and the Mediterranean basin, different studies have demonstrated the spreading of this virus in the most important producing countries including Italy, France, Spain, Greece, Germany, Switzerland, Slovakia, the Czech Republic, Austria, Croatia, Tunisia and Turkey. GLRaV-1 is broadly distributed worldwide. The genome contains ten open reading frames (ORFs) that encode ten proteins, a papain-like protease/methyl transferase/RNA helicase (ORF1a), the RNA-dependent RNA polymerase (ORF1b), p7 (ORF2), a heat shock protein 70-like protein (ORF3), p55 (ORF4), the coat protein (ORF5), the coat protein divergent 1 (ORF6), the coat protein divergent 2 (ORF7), p21 (ORF8) and p24 (ORF9). The GLRaV-1 genome consists of a positive-sense single-stranded RNA of approximately 18.7 to 18.9 kb in length that is encapsidated into filamentous particles. It was selected in this study as the preferred vector for the experiments. Planococcus citri is one of the vectors involved in GLRaV-1 transmission for which acquisition and transmission rates have been investigated under controlled conditions. GLRaV-1 is a member of the genus Ampelovirus transmitted by mealybugs ( Hemiptera: Pseudococcidae) and soft scale ( Hemiptera: Coccidae) insects. Grapevine leafroll-associated virus 1 (GLRaV-1) belongs to the GLD complex and is considered by European regulations as a pathogen that must be absent in propagative plant material. The new protocol designed has turned out to be highly sensitive as well as much more specific than the current available methods for the detection and absolute quantitation of GLRaV-1 viral titer. The method has been validated following EPPO standards and applied for the diagnosis of field plant material and transmission vectors. To this end, three new GLRaV-1 full genomes were recovered by HTS and aligned with all sequences available in the databases. The objective of this research was the development of a new GLRaV-1 detection method based on a TaqMan quantitative real-time RT-PCR targeted to the coat protein genomic region and including a host internal control in a duplex reaction. For this reason, the availability of specific, sensitive and reliable detection techniques to ascertain the sanitary status of the plants is of great importance. Grapevine leafroll-associated virus 1 (GLRaV-1) is a virus associated with grapevine leafroll disease and it is considered at the national and European level as a pathogen that must be absent in propagative plant material. Like many other crops, grapevine is susceptible to different types of diseases caused by pathogenic microorganisms. Grapevine ( Vitis vinifera L.) is one of the most important crops in the world due to its economic and social impact.
0 Comments
Leave a Reply. |
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |