植物组织和单头蚜虫中柑橘衰退病毒的快速分子检测技术

Rapid Molecular Detection Technologies of Citrus tristeza virus in Plant Tissues and Single Aphid

【目的】探寻一种快速、简便、灵敏而可靠的检测植物组织和单头蚜虫中柑橘衰退病毒的方法。【方法】运用反转录多聚酶链式反应(reverse transcription polymerase chain reaction,RT-PCR)、反转录巢式多聚酶链式反应(reverse transcription nested polymerase chain reaction,RT-nested-PCR)、印记捕获反转录巢式多聚酶链式反应(print capture reverse transcription nested polymerase chain reaction,PC-RT-nested-PCR)方法检测接种Citrus tristeza virus(CTV)四年以上的柚以及采用挤压捕获反转录巢式多聚酶链式反应(squash capture reverse transcription nested polymerase chain reaction,SC-RT-nested-PCR)检测单头蚜虫中CTV,并比较几种方法的检出率、灵敏度以及SC-RT-nested-PCR方法的有效性。【结果】对染病柚的总核酸或印迹稀释后检测,RT-PCR、RT-nested-PCR、PC-RT-nested-PCR的检测限分别为10-3、10-5和10-1倍。用质粒梯度稀释后模板进行nested-PCR检测,灵敏度达到13-14拷贝·μL-1。饲毒一周后的蚜虫经SC-RT-nested-PCR检测,除在健康琯溪蜜柚上饲喂的蚜虫未检测到目标条带外,其余在感病琯溪蜜柚上饲喂的蚜虫体内都获得132bp的目标片段。【结论】PC-RT-nested-PCR和RT-nested-PCR方法具有相同的检出率,SC-RT-nested-PCR检测单头蚜虫非常有效,PC-RT-nested-PCR和SC-RT-nested-PCR都勿需提取核酸,适用于柑橘苗木和接穗的无毒认证以及柑橘衰退病的流行监测和蚜传机理研究。

[Objective] The objective of this study was to explore a kind of quick, simple, sensitive and reliable method to detect Citrus tristeza virus in plant tissue and single aphid. [Method] RT-PCR, RT-nested-PCR and PC-RT-nested-PCR methods were used to detect CTV in pummelo inoculated CTV for at least 4 years and the positive rate and sensitivity of samples detected by these 3 methods were compared and the validity in detection of CTV in aphids was made by SC-RT-nested-PCR. [Result] Templates were used and total nucleic acid or Virus RNA obtained from imprinting after various dilutions were detected by the methods of RT-PCR, RT-nested-PCR and PC-RT-nested-PCR, and the thresholds of detection were found to be 10^-3, 10^-5 and 10^-1 folds, respectively. The sensitivity of detection of CTV by nested-PCR was up to 13-14 copies·μL^-1. Fresh viviparity （parthenogenetic） brown citrus aphids were fed on CTV infected and virus-free plants of Guanxi pummelo for a week and squashed onto Whaterman filter paper and then detected by SC-RT-nested-PCR. The 132 bp expected target PCR products were obtained in all the brown citrus aphids fed on the infected plants of Guanxi pummelo besides those fed on the virus-free plants of Guanxi pummelo. [ Conclusion ] The methods of PC-RT-nested-PCR and RT-nested-PCR showed the same positive rate of detected samples. It is efficient to detect CTV in single aphid using SC-RT-nested-PCR and there is no need to extract nucleic acid employing PC-RT-nested-PCR and SC-RT-nested-PCR methods, which are applicable to certification of virus-free citrus plants or scion as well as to the studies on monitoring and epidemiology of CTV.