title: Simultaneous detection and identification of four cherry viruses by two step multiplex RT-PCR with an internal control of plant nad5 mRNA.
abstract: A multiplex reverse transcription-polymerase chain reaction (mRT-PCR) was developed and standardized for the simultaneous detection of four cherry viruses: Cherry virus A (CVA, Genus; Capillovirus), Cherry necrotic rusty mottle virus (CNRMV, unassigned species of the Betaflexiviridae), Little cherry virus 1 (LChV-1, Genus; Closterovirus) and Prunus necrotic ringspot virus (PNRSV, Genus; Ilarvirus) with nad5 as plant internal control. A reliable and quick method for total plant RNA extraction from pome and stone fruit trees was also developed. To minimize primer dimer formation, a single antisense primer for CVA and CNRMV was used. A mixture of random hexamer and oligo (dT) primer was used for cDNA synthesis, which was highly suited and economic for multiplexing. All four viruses were detected successfully by mRT-PCR in artificially created viral RNA mixture and field samples of sweet cherry. The identity of the viruses was confirmed by sequencing. The assay could detect above viruses in diluted cDNA (10(-4)) and RNA (10(-3), except PNRSV which was detected only till ten times lesser dilution). The developed mRT-PCR will not only be useful for the detection of viruses from single or multiple infections of sweet cherry plants but also for other stone and pome fruits. The developed method will be therefore quite helpful for virus indexing, plant quarantine and certification programs. This is the first report for the simultaneous detection of four cherry viruses by mRT-PCR.
authors.affiliation: Plant Virology Lab, CSIR-Institute of Himalayan Bioresource Technology, Himachal Pradesh 176061, India.
authors.forename: Md Salik
authors.initials: MS
authors.lastname: Noorani
authors.forename: Prachi
authors.initials: P
authors.lastname: Awasthi
authors.forename: Maheshwar Prasad
authors.initials: MP
authors.lastname: Sharma
authors.forename: Raja
authors.initials: R
authors.lastname: Ram
authors.forename: Aijaz Asgar
authors.initials: AA
authors.lastname: Zaidi
authors.forename: Vipin
authors.initials: V
authors.lastname: Hallan
mesh_terms: D060885:Multiplex Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D010942:Plant Viruses / Q000235:genetics / Q000302:isolation & purification*; D027861:Prunus / Q000821:virology*; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification; D018749:RNA, Plant / Q000235:genetics / Q000302:isolation & purification; D012367:RNA, Viral / Q000235:genetics / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D017422:Sequence Analysis, DNA; D014773:Virology / Q000379:methods*
chemical_list: D012333:RNA, Messenger; D018749:RNA, Plant; D012367:RNA, Viral
keywords: CNRMV; CVA; LChV-1; Multiplex RT-PCR; PNRSV; Sweet cherry