Primers were developed which could distinguish the A and B types of Beet necrotic yellow vein virus (BNYVV) using a multiplex reverse-transcription polymerase chain reaction (mRT-PCR). RNA was extracted from 72 BNYVV isolates from Asia, Europe and North America, and the type of each isolate determined using an established single strand conformation polymorphisms (SSCP) detection method. An area of the ‘triple gene block’ region on RNA 2 was amplified and sequenced from 16 isolates of the A and B types. These sequences were aligned and two sets of PCR primers were designed to amplify unique areas common to each type. One assay produced a single 324 base-pair RT-PCR fragment from samples containing the A type and the other produced a 178 base-pair product from samples containing the B type. Fragment length differed sufficiently to allow both assays to be run in a single PCR tube. Results obtained using the new multiplex RT-PCR assay were consistent with those from the established SSCP method for all 72 reference samples.
A multiplex RT-PCR assay capable of distinguishing beet necrotic yellow vein virus type A and B / C.Ratti; G.R.G Clover; C. Rubies Autonell; V. Harju; C. M. Henry.. - In: JOURNAL OF VIROLOGICAL METHODS. - ISSN 0166-0934. - STAMPA. - 124:(2005), pp. 41-47. [10.1016/j.jviromet.2004.10.008]
A multiplex RT-PCR assay capable of distinguishing beet necrotic yellow vein virus type A and B
RATTI, CLAUDIO;RUBIES AUTONELL, CONCEPCION;
2005
Abstract
Primers were developed which could distinguish the A and B types of Beet necrotic yellow vein virus (BNYVV) using a multiplex reverse-transcription polymerase chain reaction (mRT-PCR). RNA was extracted from 72 BNYVV isolates from Asia, Europe and North America, and the type of each isolate determined using an established single strand conformation polymorphisms (SSCP) detection method. An area of the ‘triple gene block’ region on RNA 2 was amplified and sequenced from 16 isolates of the A and B types. These sequences were aligned and two sets of PCR primers were designed to amplify unique areas common to each type. One assay produced a single 324 base-pair RT-PCR fragment from samples containing the A type and the other produced a 178 base-pair product from samples containing the B type. Fragment length differed sufficiently to allow both assays to be run in a single PCR tube. Results obtained using the new multiplex RT-PCR assay were consistent with those from the established SSCP method for all 72 reference samples.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
