植物检疫

新德里番茄曲叶病毒(tomato leaf curl New Delhi virus,ToLCNDV)近两三年在很多国家开始发生扩散蔓延，是国内外高度关注的新发病毒。通过比对分析该病毒基因序列的特点，设计qPCR特异性检测引物探针ToLCNDV-173F/R/P，与引自参考文献的引物探针ToLCNDV-ToLAF/R/P、 ToLCNDV-B-F/R/P进行扩增效果的比对分析，证明了该引物探针对10种不同的阳性材料均有较好的检测效果。通过对不同病毒的阴性样品的检测试验，该方法具有较好的特异性。灵敏度分析试验显示最低可检测1.16 fg/μL病毒DNA样品，具有较高的灵敏度。建立ToLCNDV-173F/R/P qPCR定量检测的标准曲线，Ct值与模板总DNA浓度的对数值之间均呈良好的线性关系。对10种不同样品进行定量检测，植物叶片中ToLCNDV的DNA相对含量约9.93×10⁴fg/μL～2.22×10⁶fg/μL，植物种子中ToLCNDV的DNA相对含量约50.73 fg/μL～1 118.82 fg/μL。该方法克服了ToLCNDV序列变异性大、序列短小导致的引物设计困难问题，与EPPO推荐检测方法相比具有更强的灵敏度与兼容性，可以扩增该病毒的不同分离物，有效避免检测结果假阴性的出现。

Tomato leaf curl New Delhi virus(ToLCNDV) has begun to spread in many countries in the past two to three years,and is a new virus of great concern at home and abroad. By comparing and analyzing the characteristics of the gene sequence of the virus,the specific qPCR detection primer and probe ToLCNDV-173F/R/P were designed,and the amplification effect was compared with the primers,probes ToLCNDV-ToLAF/R/P and ToLCNDV-B-F/R/P quoted from the references. It is proved that the primers and probes have good detection effect for 10 different positive materials. Through the test of different virus negative samples,the method has good specificity. The sensitivity analysis test showed that the test with a minimum detectable virus DNA of 1.16 fg/μL had a high sensitivity. The standard curve of ToLCNDV-173 F/R/P q PCR quantitative detection was established,and the correlation between Ct value and logarithmic value of template total DNA concentration was linear. Through quantitative testing of 10 different samples,the DNA relative content of ToLCNDV in leaves was about 9.93 ×10⁴fg/μL-2.22 ×10⁶fg/μL,and the DNA relative content of ToLCNDV in seeds was about 50.73 fg/μL-1 118.82 fg/μL. This method overcomes the difficulty of primer design due to the large variability and shortness of ToLCNDV sequence. Compared with the EPPO recommended detection methods,this method has stronger sensitivity and compatibility,and can easily amplify different isolates of the virus,effectively avoiding the appearance of false negative detection results.