福建农业学报2018，Vol.33Issue(3)：288-292,5.DOI:10.19303/j.issn.1008-0384.2018.03.013
太子参BBWV2和TuMV的巢式RT-PCR检测
Nested RT-pCR Assay for Detecting Broad bean wilt virus 2 and Turnip mosaic virus in Pseudostellaria heterophylla
摘要
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 l05 x 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.关键词
太子参/太子参蚕豆萎蔫病毒2 (BBWV2)/芜菁花叶病毒(TuMV)/巢式RT-PCRKey words
Pseudostellaria heterophylla/Broad bean wilt virus 2 (BBWV2)/Turnip mosaic virus (TuMV)/nested RT-PCR分类
农业科技引用本文复制引用
匡云波,侯令,方晓勤,王佳璐,杨燕君,叶祖云..太子参BBWV2和TuMV的巢式RT-PCR检测[J].福建农业学报,2018,33(3):288-292,5.基金项目
福建省自然科学基金项目(2017J05058) （2017J05058）
福建省教育厅科技扶贫产学研专项(JZ160394) （JZ160394）
国家中药标准化项目(ZYBZH-Y-FJ-08) （ZYBZH-Y-FJ-08）
