Nested RT-PCR Assay for Detecting Broad bean wilt virus 2 and Turnip mosaic virus in Pseudostellaria heterophylla
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摘要: 为探求快速、灵敏的太子参脱毒检测技术，建立太子参蚕豆萎蔫病毒2（Broad bean wilt virus 2，BBWV2）和芜菁花叶病毒（Turnip mosaic virus，TuMV）的巢式RT-PCR快速检测方法，根据GenBank数据库中这2种病毒外壳蛋白（coat protein，cp）基因核苷酸序列的保守区域分别设计2对简并引物，建立和优化巢式RT-PCR扩增体系，继而进行常规RT-PCR和巢式RT-PCR灵敏度的比较，并应用巢式RT-PCR对7份田间栽培太子参病样和4份脱毒苗样品进行检测。结果显示：BBWV2、TuMV的巢式引物最佳退火温度分别为60、62℃；采用巢式RT-PCR，这两种病毒样品cDNA原液在稀释105倍后仍能扩增出特异性条带，比常规RT-PCR的灵敏度至少高10倍。本研究建立的巢式RT-PCR检测方法可快速、稳定、准确地检测BBWV2和TuMV，且具有更高的灵敏度，更能满足太子参脱毒检测的需要。
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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-PCR
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. -
图 1 不同退火温度下BBWV2和TuMV的巢式RT-PCR扩增
注：A为BBWV2；B为TuMV；M为DM2000；1～6依次为52、54、56、58、60、62℃。
Figure 1. Amplification of BBWV2 and TuMV by nested RT-PCR at different annealing temperatures
图 2 常规RT-PCR扩增的灵敏度分析
注：A为BBWV2；B为TuMV；M为DM2000；1～5依次为cDNA原液稀释101、102、103、104、105倍；6为阴性对照。
Figure 2. Amplification sensitivity of conventional RT-PCR
图 3 巢式RT-PCR扩增的灵敏度分析
注：A为BBWV2；B为TuMV；1为阴性对照；2～7依次为cDNA原液稀释101、102、103、104、105、106倍；M为DM2000。
Figure 3. Amplification sensitivity of nested RT-PCR
图 4 太子参样品的巢式RT-PCR病毒检测结果
注：A为BBWV2；B为TuMV；1为阴性对照；2～4为福建病样；5～8依次为江苏、湖南、贵州和山东病样；M为DM2000；9～11为种胚脱毒的组培苗；12为茎尖脱毒的组培苗。
Figure 4. Detection of BBWV2 and TuMV in P.heterophylla samples by nested RT-PCR
表 1 BBWV2和TuMV的巢氏RT-PCR检测引物
Table 1. Nested RT-PCR primers for BBWV2 and TuMV identification
引物名称 引物序列(5′-3′) 目的片段大小
/bp退火温度
/℃BBWV2--F TTGGGHTCWAGYYTGGGACGYTTRT 1345 57 BBWV2-R TTRTARAACTTCTTGCTCCCACGM BBWV2-NF AAYGCTCCRAARGTVGATGCYARRA 498 - BBWV2-NR CBCCTCTTGCYGARTCYAAATCCCA TuMV-F AGGTGAAAYGCTTGATGCAGGTY 775 60 TuMV-R GTTHCCATCCARKCCGAACAAAT TuMV-NF TCATCTRATYCTRTACACGCCRGAGC 337 - TuMV-NR TGTATGGTCGGTCTTGGTTACGC 
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