Nested RT-PCR Assay for Detecting Broad bean wilt virus 2 and Turnip mosaic virus in Pseudostellaria heterophylla

KUANG Yun-bo; HOU Ling; FANG Xiao-qin; WANG Jia-lu; YANG Yan-jun; YE Zu-yun

doi:10.19303/j.issn.1008-0384.2018.03.013

Volume 33 Issue 3

Mar. 2019

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Article Navigation > Fujian Journal of Agricultural Sciences > 2018 > 33(3): 288-292

KUANG Yun-bo, HOU Ling, FANG Xiao-qin, WANG Jia-lu, YANG Yan-jun, YE Zu-yun. Nested RT-PCR Assay for Detecting Broad bean wilt virus 2 and Turnip mosaic virus in Pseudostellaria heterophylla[J]. Fujian Journal of Agricultural Sciences, 2018, 33(3): 288-292. doi: 10.19303/j.issn.1008-0384.2018.03.013

Citation:

KUANG Yun-bo, HOU Ling, FANG Xiao-qin, WANG Jia-lu, YANG Yan-jun, YE Zu-yun. Nested RT-PCR Assay for Detecting Broad bean wilt virus 2 and Turnip mosaic virus in Pseudostellaria heterophylla[J]. Fujian Journal of Agricultural Sciences, 2018, 33(3): 288-292. doi: 10.19303/j.issn.1008-0384.2018.03.013

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Nested RT-PCR Assay for Detecting Broad bean wilt virus 2 and Turnip mosaic virus in Pseudostellaria heterophylla

doi: 10.19303/j.issn.1008-0384.2018.03.013

1.
College of Life Sciences, Ningde Normal University, Ningde, Fujian 352100, China
2.
Engineering Research Center for Medical Plants with Fujian Provincial Characteristics, Ningde, Fujian 352100, China

Received Date: 2018-01-09
Rev Recd Date: 2018-02-16
Publish Date: 2018-03-01

Abstract

Abstract

To enable a rapid and sensitive determination of being virus-free for a Pseudostellaria heterophylla seedling, a nested RT-PCR method was developed. Detection of the presence of either Broad bean wilt virus(BBWV2) or Turnip mosaic virus(TuMV) in the sample was a proof of the existence of the diseases. Two pairs of degenerate primers were designed respectively according to the conserved sequence regions of the two coat protein (cp) genes of the viruses from GenBank. The nested RT-PCR detection protocols were optimized. Subsequently, detecting sensitivities of the conventional and the nested RT-PCRs were compared. Meanwhile, 7 samples of cultivated P. heterophylla along with 4 of virus-free test-tube plantlets were used in a challenge test on the nested RT-PCR methodology. The results showed that, by using the annealing temperature of 60℃ for BBWV2 and 62℃ for TuMV on the nested primers, specific bands could be detected on a 10⁵x dilution of the first strand cDNA for both viruses. That suggested at least 10-fold higher a sensitivity was achieved by the nested than the conventional RT-PCR. It was concluded that the newly established assay method was rapid, stable, sensitive and accurate for detecting BBWV2 or TuMV in P. heterophylla, and therefore, could be adequately applied to certify a virus-free status or not on a P. heterophylla sample.
- Pseudostellaria heterophylla,
- Broad bean wilt virus 2(BBWV2),
- Turnip mosaic virus(TuMV),
- nested RT-PCR

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References(27)

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