柑橘碎叶病毒巢式RT-PCR检测方法建立及应用

Establishment of nested RT-PCR for detecting Citrus tatter leaf virus

柑橘碎叶病毒(Citrus tatter leaf virus,CTLV)是威胁柑橘生产的重要病原之一。本研究在柑橘碎叶病毒一步法RT-PCR检测体系的基础上建立了CTLV的巢式RT-PCR检测方法,并对其检测灵敏度及特异性进行了分析。结果表明,该方法检测灵敏度比一步法RT-PCR至少提高100倍,检测模板总核酸的最低浓度约1.27μg·L-1,具有良好的特异性。在对142个田间样品、24个茎尖苗及20个接毒的腊斯克枳橙的检测中,巢式RT-PCR的CTLV检出率最高,为21.5%,比半巢式RT-PCR检出率高3.2%,比一步法RT-PCR检出率高2.1%。该方法灵敏度高,特异性强,适用于对无病毒苗木生产的监控和田间样品的快速检测。

Citrus tatter leaf virus（CTLV） is an economically important pathogen of citrus.To establish a sensitive and specific method for detection of this virus,a nested RT-PCR was developed by comparing the detection efficiency of five primer sets.Total nucleotide acid extraction of CTLV-inoculated samples were serially tenfold diluted to evaluate the sensitivity,and samples infected with CTV,SDV,HLB,Canker,or CEVd were used to determine the specificity.The results showed that the lowest detection limit of the total nucleic acid by the method reached about 1.27 μg·L-1,which was hundred times lower than that of One-step RT-PCR assay at least.No positive result was obtained from the samples without CTLV using this method.In the detection of 142 samples collected from orchards,24 shoot-tip grafting citrus,and 20 rusk citrange infected by CTLV,the positive rates using the nested RT-PCR,the routine semi-nested PCR,and the one-step RT-PCR were 21.5%,18.3% and 19.4% respectively.It suggested that the nested RT-PCR is a sensitive,specific method and can be used as a useful tool for CTLV detection.