Simultaneous detection and differentiation of three genotypes of Brassica yellows virus by multiplex reverse transcription-polymerase chain reaction

Abstract

BackgroundBrassica yellows virus (BrYV), proposed to be a new polerovirus species, three distinct genotypes (BrYV-A, BrYV-B and BrYV-C) have been described. This study was to develop a simple, rapid, sensitive, cost-effective method for simultaneous detection and differentiation of three genotypes of BrYV.ResultsIn this study, a multiplex reverse transcription-polymerase chain reaction (mRT-PCR) was developed for simultaneous detection and differentiation of the three genotypes of BrYV. The three genotypes of BrYV and Tunip yellows virus (TuYV) could be differentiated simultaneously using six optimized specific oligonucleotide primers, including one universal primer for detecting BrYV, three BrYV genotype-specific primers, and a pair of primers for specific detection of TuYV. Primers were designed from conserved regions of each virus and their specificity was confirmed by sequencing PCR products. The mRT-PCR products were 278 bp for BrYV-A, 674 bp for BrYV-B, 505 bp for BrYV-C, and 205 bp for TuYV. Amplification of three target genotypes was optimized by increasing the PCR annealing temperatures to 62 °C. One to three fragments specific for the virus genotypes were simultaneously amplified from infected samples and identified by their specific molecular sizes in agarose gel electrophoresis. No specific products could be amplified from cDNAs of other viruses which could infect crucifer crops. Detection limits of the plasmids for multiplex PCR were 100 fg for BrYV-A and BrYV-B, 10 pg for BrYV-C, and 1 pg for TuYV, respectively. The mRT-PCR was applied successfully for detection of three BrYV genotypes from field samples collected in China.ConclusionsThe simple, rapid, sensitive, and cost-effective mRT-PCR was developed successfully for detection and differentiation of the three genotypes of BrYV.

A Simple Method for the Acquisition and Transmission of Brassica Yellows Virus from Transgenic Plants and Frozen Infected Leaves by Aphids

“…Brassica yellows virus (BrYV) is a tentative newly identified species in the genus of Polerovirus , and it is closely related to turnip yellows virus (TuYV) [ 8 , 9 ]. It is spread widely throughout China as well as in South Korea and Japan [ 4 , 10 , 11 ]. BrYV has a wide host range, although it mainly infects Brassicaceae crops, including cabbage ( Brassica oleracea var.…”

“…botrytis ), mustard ( B. juncea ), and turnip ( Raphanus sativus var. oleifera ), usually causing leaf malformations and yellowing in the field [ 4 , 9 , 10 , 11 , 12 ]. BrYV has at least three genotypes (BrYV-A, B, and C) as determined by sequence analyses, and full-length infectious cDNA clones of these three genotypes have been developed successfully [ 8 , 10 , 11 , 13 , 14 ].…”

“…oleifera ), usually causing leaf malformations and yellowing in the field [ 4 , 9 , 10 , 11 , 12 ]. BrYV has at least three genotypes (BrYV-A, B, and C) as determined by sequence analyses, and full-length infectious cDNA clones of these three genotypes have been developed successfully [ 8 , 10 , 11 , 13 , 14 ]. The full-length amplicon of BrYV-C has been successfully transferred into Arabidopsis thaliana , resulting in two stable transgenic lines, 111 and 412, which exhibit severe symptoms, including dwarfism and purple leaves [ 15 ].…”

Viruses infecting cool season crops in the northern Turkey

“…In terms of host range, CaMV ranks second only to TuMV, based on the number of susceptible hosts among vegetable brassicas (Spence et al 2007). CMV has extremely wide host range, and is one of the main pathogens of cruciferous crops (Zhang et al 2016). CMV is efficiently transmitted by more than 75 species of aphids in non-persistent manner (Krenz et al 2015).…”

A Simple Method for Acquisition and Inoculation of Brassica Yellows Virus from Transgenic Plants and Frozen Infected Leaves by Aphids

“…genotypes (BrYV-A, B, and C) according to sequence analysis, the full-length infectious cDNA clones of these three genotypes have been developed successfully [8,[10][11][12][13]. Previously work have shown that the full-length amplicon of the virus has been successfully transferred into Arabidopsis thaliana and obtained stable genetically transgenic lines named 111 and 412 which all exhibited severe symptoms including dwarfing and the purple leaves [14].…”