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

View Article: PubMed Central - PubMed

ABSTRACT

Background: Brassica 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.

Results: In 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.

Conclusions: The simple, rapid, sensitive, and cost-effective mRT-PCR was developed successfully for detection and differentiation of the three genotypes of BrYV.

No MeSH data available.


Agarose gel electrophoresis of 6 field samples collected from Haidian District of Beijing City by multiplex RT-PCR. Lanes 1–6, different crucifer leaf tissue samples collected from different fields in Haidian District of Beijing City, Lane 1, Raphanus sativus L.; Lane 2, Brassica oleracea var. acephala f.tricolor; Lane 3, Brassica oleracea L. var. botrytis L.; Lane 4, Brassica rapa pekinensis; Lane 5, Brassica oleracea L. var. Capitata f. rubra DC.; Lane 6, Brassica oleracea L. var. caulorapa DC.; Lane 7, negative control, the healthy crucifer plant sample; Lane 8, positive control, BrYV-A, -B and -C co-infected Nicotiana benthamiana
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Fig4: Agarose gel electrophoresis of 6 field samples collected from Haidian District of Beijing City by multiplex RT-PCR. Lanes 1–6, different crucifer leaf tissue samples collected from different fields in Haidian District of Beijing City, Lane 1, Raphanus sativus L.; Lane 2, Brassica oleracea var. acephala f.tricolor; Lane 3, Brassica oleracea L. var. botrytis L.; Lane 4, Brassica rapa pekinensis; Lane 5, Brassica oleracea L. var. Capitata f. rubra DC.; Lane 6, Brassica oleracea L. var. caulorapa DC.; Lane 7, negative control, the healthy crucifer plant sample; Lane 8, positive control, BrYV-A, -B and -C co-infected Nicotiana benthamiana

Mentions: To evaluate the feasibility of multiplex RT-PCR for the diagnosis of field samples, 6 crucifer leaf tissue samples collected from different fields in Haidian District of Beijing City were used to test and standardize the mRT-PCR. One or two genotypes of BrYV could be detected by the mRT-PCR. The total RNA of BrYV-A, -B and -C agro-infected N. benthamiana served as positive control and it could produce three distinct fragments, 278 bp, 674 bp and 505 bp, respectively (Fig. 4). The healthy plant samples served as negative control gave no signal as expected. Following gel electrophoresis, the bands of PCR products were clear and there were no discernible differences in the size of the virus amplicons obtained from the different field samples indicating that the multiplex PCR assay could be used to detect the three genotypes of BrYV from a wide range of field areas. And the detection results were further confirmed by sequencing PCR products. With the high sensitivity and specificity of the mRT-PCR method, it is easy to detect and differentiate the three genotypes of BrYV.Fig. 4


Simultaneous detection and differentiation of three genotypes of Brassica yellows virus by multiplex reverse transcription-polymerase chain reaction
Agarose gel electrophoresis of 6 field samples collected from Haidian District of Beijing City by multiplex RT-PCR. Lanes 1–6, different crucifer leaf tissue samples collected from different fields in Haidian District of Beijing City, Lane 1, Raphanus sativus L.; Lane 2, Brassica oleracea var. acephala f.tricolor; Lane 3, Brassica oleracea L. var. botrytis L.; Lane 4, Brassica rapa pekinensis; Lane 5, Brassica oleracea L. var. Capitata f. rubra DC.; Lane 6, Brassica oleracea L. var. caulorapa DC.; Lane 7, negative control, the healthy crucifer plant sample; Lane 8, positive control, BrYV-A, -B and -C co-infected Nicotiana benthamiana
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC5120529&req=5

Fig4: Agarose gel electrophoresis of 6 field samples collected from Haidian District of Beijing City by multiplex RT-PCR. Lanes 1–6, different crucifer leaf tissue samples collected from different fields in Haidian District of Beijing City, Lane 1, Raphanus sativus L.; Lane 2, Brassica oleracea var. acephala f.tricolor; Lane 3, Brassica oleracea L. var. botrytis L.; Lane 4, Brassica rapa pekinensis; Lane 5, Brassica oleracea L. var. Capitata f. rubra DC.; Lane 6, Brassica oleracea L. var. caulorapa DC.; Lane 7, negative control, the healthy crucifer plant sample; Lane 8, positive control, BrYV-A, -B and -C co-infected Nicotiana benthamiana
Mentions: To evaluate the feasibility of multiplex RT-PCR for the diagnosis of field samples, 6 crucifer leaf tissue samples collected from different fields in Haidian District of Beijing City were used to test and standardize the mRT-PCR. One or two genotypes of BrYV could be detected by the mRT-PCR. The total RNA of BrYV-A, -B and -C agro-infected N. benthamiana served as positive control and it could produce three distinct fragments, 278 bp, 674 bp and 505 bp, respectively (Fig. 4). The healthy plant samples served as negative control gave no signal as expected. Following gel electrophoresis, the bands of PCR products were clear and there were no discernible differences in the size of the virus amplicons obtained from the different field samples indicating that the multiplex PCR assay could be used to detect the three genotypes of BrYV from a wide range of field areas. And the detection results were further confirmed by sequencing PCR products. With the high sensitivity and specificity of the mRT-PCR method, it is easy to detect and differentiate the three genotypes of BrYV.Fig. 4

View Article: PubMed Central - PubMed

ABSTRACT

Background: Brassica 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.

Results: In 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.

Conclusions: The simple, rapid, sensitive, and cost-effective mRT-PCR was developed successfully for detection and differentiation of the three genotypes of BrYV.

No MeSH data available.