A Multiplex RT-PCR Assay for Detecting Three Pathogens Infecting Citrus Plants
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摘要:
目的 建立柑橘黄化脉明病毒（citrus yellow vein clearing virus, CYVCV）、柑橘衰退病毒（citrus tristeza virus, CTV）和啤酒花矮化类病毒（hop stunt viroid, HSVd）的多重RT-PCR检测体系。 方法 设计多重RT-PCR引物，分析其特异性，确定其最佳浓度比、最适退火温度及灵敏度，在此基础上对福建地区的柑橘样品进行检测。 结果 确定了CYVCV-F/R、CTV-F/R和HSVd-F/R等3对引物的最佳浓度比例为1∶1∶2，最适退火温度为52.9 ℃，灵敏度结果显示该体系可检测模板稀释到10−2的阳性样品。应用该体系对采自福建部分地区的157份柑橘样品进行检测，结果发现，CYVCV、CTV和HSVd的检出率分别为47.1%、56.7%和22.9%。 结论 成功建立了柑橘CYVCV、CTV和HSVd病原的多重RT-PCR检测方法，为该类病害的检测提供准确、快速的检测方法。 Abstract:Objective A multiplex RT-PCR assay to simultaneously detect citrus yellow vein clearing virus (CYVCV), citrus tristeza virus (CTV), and hop stunt viroid (HSVd) was developed. Method Multiplex RT-PCR primers were designed, and their specificity was analyzed. The optimal concentration ratio, annealing temperature and sensitivity of primers were determined. Moreover, citrus samples from Fujian region were detected by the established multiplex RT-PCR. Result The optimized concentration ratio of CYVCV-F/R, CTV-F/R, and HSVd-F/R primers was 1:1:2, and the annealing temperature at 52.9 ℃ for the assay. The assay displayed a sensitivity at 10-2 dilution and the positive detections on CYVCV of 47.1%, on CTV of 56.7%, and on HSVd of 22.9% on 157 detected samples. Conclusion A rapid, accurate, applicable multiplex RT-PCR method for CYVCV, CTV, and HSVd detections was successfully established. -
图 1 单重RT-PCR扩增结果
M：D2000Plus DNA分子量标准；1：CYVCV-F/R扩增；2：CTV-F/R扩增；3：HSVd-F/R扩增。
Figure 1. Amplification of single RT-PCR
M: D2000Plus DNA marker; 1: amplified by CYVCV-F/R; 2: amplified by CTV-F/R; 3: amplified by HSVd-F/R.
图 2 不同引物浓度组合的扩增结果
M：D2000Plus DNA分子量标准，1～8引物浓度组合参见表1。
Figure 2. Amplification of varied concentration ratios of combined primers
M: D2000Plus DNA marker, 1–8: concentration ratio of combined primers as shown on Table 1.
图 4 单重及多重RT-PCR灵敏度检测结果
M：D2000Plus DNA分子量标准，1：100，2：10−1，3：10−2，4：10−3，5：10−4，6：10−5，7：10−6，8：阴性对照。
Figure 4. Assay sensitivity of single and multiplex RT-PCR
M: D2000Plus DNA marker; 1: 100; 2: 10−1; 3: 10−2; 4: 10−3; 5: 10−4; 6: 10−5; 7: 10−6; 8: negative control.
图 5 多重RT-PCR体系的应用
A： 多重RT-PCR检测样品； M：D2000Plus DNA分子量标准，1～16：16份柑橘样品，17：阴性对照。B： 检出率分析； C： 复合侵染分析。
Figure 5. Application of multiplex RT-PCR assay
A: Detection by multiplex RT-PCR; M: D2000Plus DNA marker; 1–16: 16 citrus samples; 17: negative control; B: detection rate; C: detection on mixed infections.
表 1 多重RT-PCR引物浓度组合
Table 1. Primers for multiplex RT-PCR assay
（单位：μmol·L−1） 引物名称
Primer name引物浓度组合
Combinations of different primer concentration1 2 3 4 5 6 7 8 CYVCV-F/R 0.2 0.2 0.15 0.15 0.2 0.15 0.15 0.15 CTV-F/R 0.2 0.1 0.15 0.15 0.15 0.15 0.2 0.2 HSVd-F/R 0.3 0.15 0.3 0.2 0.3 0.15 0.3 0.2 
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