Abstract:

【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.

Key words: Citrus tristeza virus, molecular detection, plant tissues, single aphid