Sequence‐independent Amplification with Genome Multiplexing to Establish Complete Genome of Multipartite RNA Viruses: Cucumber mosaic virus as a Case Study. Issue 6 (18th April 2017)

• Record Type: Journal Article
• Authors: Kumar, Surender; Kumari, Reenu; Hallan, Vipin
• Is Part Of: Journal of phytopathology. Volume 165:Issue 6(2017:Jun.)
• Page Start: 361
• Page End: 366
• Publication Date: 2017-04-18
• Subjects: dsRNA and genome multiplexing -- sequence‐independent single‐primer amplification (SISPA) -- SISPA for genome multiplexing; Plant diseases -- Periodicals; 632
• DOI: 10.1111/jph.12569
• Languages: English
• ISSNs: 0931-1785

Abstract

Abstract: Reverse transcription–polymerase chain reaction (RT‐PCR) is considered one of the accurate and reliable techniques for plant virus detection and genome characterization. Environmental conditions, error prone replication and biological competition lead to mutations and sequence variation. This results in generation of various strains/variants. These conditions present difficulties in genome amplification, especially with techniques which utilize prior sequence information, such as RT‐PCR. Keeping in view this difficulty, in this study, we used replicating forms (RFs) from ( Cucumber mosaic virus ) infected Nicotiana tabacum var . Xanthi as a precursor for ligation of self‐primed anchor primer at 3′‐hydroxyl end and subsequently used it for first strand cDNA synthesis. This cDNA was amplified further by sequence‐independent single‐primer amplification (SISPA) for genome multiplexing of CMV, a tripartite ssRNA positive‐sense virus. It was also observed that the presence of anchor primer aids towards cloning of genome segments. These results demonstrate genome multiplexing for complete genome characterization of single‐stranded, multipartite plant RNA viruses as the new application of SISPA.