松材线虫微滴式数字PCR检测体系的建立

Establishment of droplet digital PCR system for detection of Bursaphelenchus xylophilus

【目的】建立松材线虫微滴式数字PCR检测体系,验证该检测体系的有效性,为松材线虫检测提供一个更精确灵敏的定性检测方法。【方法】基于松材线虫16S rDNA基因序列,使用在线工具设计特异性引物和探针。优化PCR反应条件,明确微滴式数字PCR的最佳反应条件。再以采集自不同地区的6株松材线虫和12株拟松材线虫、滑刃线虫、伞滑刃线虫、垫刃线虫的基因组DNA溶液为模板对该体系的特异性进行检测;以稀释10倍的标准DNA为模板,设置不同浓度梯度来验证该体系的灵敏度;以3个不同浓度的DNA标准品稀释模板检测微滴式数字PCR的可重复性。最后以人工模拟添加线虫样品和感病松木样品DNA提取液为模板对该体系的实用性进行验证。【结果】①设计了松材线虫特异性引物探针一组,引物:P1904-F、P2057-R;探针:Probe-1938。②PCR反应体系经优化,确定其最佳退火温度为55℃。③特异性验证表明,设计的引物探针可以将松材线虫与其他相近种的线虫区分开。④灵敏度检测结果发现,该检测体系的检出浓度在0.51~20000 copies/μL。⑤对3组不同浓度标准样品的可重复性检测发现,该检测体系的变异系数在9.12%~0.31%,并且样品浓度与变异系数呈负相关。⑥使用该检测体系检测林间感病松木样品和人工模拟加入松材线虫的样品发现,该检测体系能够特异地检测出样品中的松材线虫,且拷贝数与样品中的线虫量呈正向相关。【结论】本研究建立了松材线虫微滴式数字PCR检测体系,该方法特异性好,检测灵敏度高,变异系数小,可高效地检测出样品中的松材线虫。该体系的建立可为松材线虫病理学和生态学研究提供可靠的研究数据。

【Objective】The study aimed to establish droplet digital detection system and verify the effectiveness of it,in order to provide a more accurate and sensitive method for the detection of Bursaphelenchus xylophilus.【Method】Genus-specific primers and probe were designed based on 16 S rDNA gene sequence in using online tools for primers and probe design.Optimize the PCR reaction conditions and determine the best reaction conditions of droplet digital detection system.The specificity of this system was detected by using the genomic DNA solutions of 6 strains of B.xylophilus and 12 strains of B.mucronatus,B.thailandae,B.sexdentati,B.dalianensis,Aphelenchoides parasaprophilus,A.resinosi and Cylindrotylenchus pini.The sensitivity of this system was verified by setting different concentration gradients with 10 times diluted standard DNA as a template.The repeatability of droplet digital PCR was detected by three different concentrations of DNA standard dilution templates.Finally,the practicability of this system was verified by using the DNA extract of nematode samples and susceptible pine samples as templates.【Result】(ⅰ)A set of pine wood nematode specific primers and probe were designed which named primers:P1904-F,P2057-R;Probe:Probe-1938.(ⅱ)The optimize annealing temperature of PCR reaction system was determined to be 55℃.(ⅲ)The Specificity verification showed that the designed primers and probe could distinguish the B.xylophilus from other similar nematodes.(ⅳ)The results of sensitivity detection showed that the detection concentration of this detection system was between 0.51-20000 copies/μL.(ⅴ)The repeatability test of three groups of standard samples with different concentrations found that the coefficient of variation of this detection system was between 0.31%-9.12%,and the sample concentration was negatively correlated with the coefficient of variation.(ⅵ)Dd-PCR were used to test the wood sample which collected from affected trees and simulation add B.xylophilus,the results has shown that this method can detect B.xylophilus in the samples,moreover,the copy number was positively correlated with the amount of nematodes in the sample.【Conclusion】In this study,the dd-PCR detection system of pine wood nematode was established.This method exhibit high specificity,high sensitivity,low coefficient of variation,and it can detect B.xylophilus in wood samples with high efficiency.The establishment of this system could provide reliable data for the study of pathology and ecology of B.xylophilus.