核酸共提取试剂盒应用于柑橘危险性病害检测的效率评价

Evaluation of the efficiency of nucleic acid co-extraction kit applied to the detection of dangerous diseases of citrus

柑橘易受到不同遗传类型病原的复合感染,传统病原检测方法需单一核酸提取,使得检测步骤繁琐,影响检测效率。以普通单一核酸提取试剂盒为对照,采用核酸共提取试剂盒,通过分析核酸完整性和浓度以及病原检测效率,以期探讨核酸共提取试剂盒应用于柑橘危险性病害检测的可行性,从而简化柑橘病原检测方法的核酸提取步骤。结果表明,相比普通RNA提取试剂盒,核酸共提取试剂盒提取RNA的完整性、纯度、浓度以及柑橘衰退病毒病原检测效率均无显著差异,可达到相同检测效果。相比普通DNA提取试剂盒,核酸共提取试剂盒提取DNA的完整性、纯度均无显著性差异,但其浓度显著降低,使得柑橘黄龙病和柑橘溃疡病病原检测效率也显著降低;通过增加PCR反应的起始模板量,核酸共提取试剂盒检测柑橘黄龙病和柑橘溃疡病病原的效率与普通DNA试剂盒无显著差异,可达到相同检测效果。综上所述,核酸共提取试剂盒可应用于柑橘危险性病害检测,将有助于提高病原复合感染样品的检测效率。

Citrus is easy to be mixed-infected by pathogens with different genetic types.Traditional pathogen detection methods require the extraction of single nucleic acid,which makes the detection procedure complicated and affects the detection efficiency.In this paper,comparing with the common single nucleic acid extraction kit,the nucleic acid co-extraction kit is evaluated in terms of integrity and concentration of nucleic acid as well as the efficiency of pathogen detection,in order to explore its feasibility in the detection of citrus dangerous diseases,and overcome the shortcoming of traditional pathogen detection methods.The results showed that compared to common RNA extraction kit,there were no significant differences in the integrity,purity,concentration of RNA,and Citrus tristeza virus detection efficiency of RNA extracted by the nucleic acid co-extraction kit,and the same detection effect could be achieved.Compared with the common DNA extraction kit,the integrity and purity of DNA extracted by the nucleic acid co-extraction kit were not significantly different,but the concentration was significantly reduced,which significant decrease the efficiency of DNA-type viruses.By increasing the template amount of PCR reaction,the efficiency of nucleic acid co-extraction kit for Citrus Huanglongbing and Xanthomonas citri subsp.citri was not significantly different from the common DNA kit,and the same detection effect could be achieved.In conclusion,the nucleic acid co-extraction kit could be applied to the detection of citrus dangerous diseases,and would be helpful to improve the detection efficiency of multiply pathogens in one sample.