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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.


Detection limit of multiplex RT-PCR using ten-fold serial dilutions of individually plasmids. (a, b, c, e), uniplex PCR; (d, f), multiplex PCR. Lane M, 100 bp DNA marker. Lanes 1–8, 10 ng, 1 ng, 100 pg, 10 pg, 1 pg, 100 fg, 10 fg, and 1 fg of plasmids of different BrYV genotypes. (e), pMD19-TuYV-P0 and pMD19-56#-1 were used for dectection of TuYV
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Fig2: Detection limit of multiplex RT-PCR using ten-fold serial dilutions of individually plasmids. (a, b, c, e), uniplex PCR; (d, f), multiplex PCR. Lane M, 100 bp DNA marker. Lanes 1–8, 10 ng, 1 ng, 100 pg, 10 pg, 1 pg, 100 fg, 10 fg, and 1 fg of plasmids of different BrYV genotypes. (e), pMD19-TuYV-P0 and pMD19-56#-1 were used for dectection of TuYV

Mentions: To compare the sensitivity of multiplex RT-PCR versus uniplex RT-PCR, 10-fold serial dilutions of the purified plasmids pCaBrA, pCaBrB, pCaBrC, pMD19-TuYV-P0, and pMD19-56#-1 were used simultaneously as templates in uniplex RT-PCR and multiplex RT-PCR reactions. The results showed that detection limits of the DNA quantity for multiplex RT-PCR were 100 fg for BrYV-A and BrYV-B, 10 pg for BrYV-C, and 1 pg for TuYV, respectively, if the concentration of the plasmids was similar (Fig. 2).Fig. 2


Simultaneous detection and differentiation of three genotypes of Brassica yellows virus by multiplex reverse transcription-polymerase chain reaction
Detection limit of multiplex RT-PCR using ten-fold serial dilutions of individually plasmids. (a, b, c, e), uniplex PCR; (d, f), multiplex PCR. Lane M, 100 bp DNA marker. Lanes 1–8, 10 ng, 1 ng, 100 pg, 10 pg, 1 pg, 100 fg, 10 fg, and 1 fg of plasmids of different BrYV genotypes. (e), pMD19-TuYV-P0 and pMD19-56#-1 were used for dectection of TuYV
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig2: Detection limit of multiplex RT-PCR using ten-fold serial dilutions of individually plasmids. (a, b, c, e), uniplex PCR; (d, f), multiplex PCR. Lane M, 100 bp DNA marker. Lanes 1–8, 10 ng, 1 ng, 100 pg, 10 pg, 1 pg, 100 fg, 10 fg, and 1 fg of plasmids of different BrYV genotypes. (e), pMD19-TuYV-P0 and pMD19-56#-1 were used for dectection of TuYV
Mentions: To compare the sensitivity of multiplex RT-PCR versus uniplex RT-PCR, 10-fold serial dilutions of the purified plasmids pCaBrA, pCaBrB, pCaBrC, pMD19-TuYV-P0, and pMD19-56#-1 were used simultaneously as templates in uniplex RT-PCR and multiplex RT-PCR reactions. The results showed that detection limits of the DNA quantity for multiplex RT-PCR were 100 fg for BrYV-A and BrYV-B, 10 pg for BrYV-C, and 1 pg for TuYV, respectively, if the concentration of the plasmids was similar (Fig. 2).Fig. 2

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.