柑橘衰退病毒RT-RPA-LFD可视化检测方法的建立及应用

Establishment and application of RT-RPA-LFD visualization assay for rapid detection of citrus tristeza virus

【目的】柑橘衰退病由柑橘衰退病毒(citrus tristeza virus,CTV)引起,是一种世界性的重要柑橘病害。为实现CTV的田间快速检测,建立一种准确、快速且可视化的检测方法。【方法】以CTV外壳蛋白(CP)的保守区域为靶标,设计3对特异性引物和探针,通过引物筛选,以及优化引物浓度、反应时间和反应温度等条件,建立CTV的反转录-重组酶聚合酶扩增-侧流层析试纸条(RT-RPA-LFD)快速检测方法,明确其灵敏度,并用于田间疑似样品的检测。【结果】建立了CTV的RT-RPA-LFD检测方法:最佳检测引物为RPA-1F/R,对应探针为RPA-P,最佳反应条件为40℃,25 min,且与其他5种柑橘病毒无交叉反应。该方法的灵敏度是RT-PCR的100倍,最低可检测到2.12×10^(1)拷贝·μL^(-1)的CTV核酸,与RT-qPCR相当。采用RT-RPA-LFD法在67份田间样品中检测出CTV阳性样品41份,与RT-PCR法检测结果一致。【结论】建立的CTV RT-RPA-LFD法具有操作简单、快速、结果可视等优点,适合基层植保工作者对田间样品开展快速检测。

【Objective】Tristeza caused by citrus tristeza virus(CTV)is one of the most destructive cit-rus diseases in the world,which is mainly spread by several aphid species and bud-grafting.Severe CTV isolates could cause quick decline of sour orange rootstock,and stem pitting of susceptible culti-vars.In recent years,stunted,severe stem pitting and reduced fruit quality were observed in Newhall na-vel orange and some tangor cultivars,causing severe economic losses in major citrus-growing provinc-es of China,especially in Hunan,Jiangxi,Yunnan,Sichuan provinces.Prompt and accurate CTV detec-tion in the nursery and field samples is necessary to control CTV.To date,serological techniques,re-verse transcription PCR(RT-PCR),RT-real-time PCR(RT-qPCR)and other methods have been used to detect CTV.However,these traditional detection techniques are generally flawed.The purpose of this study was to establish a reliable,accurate,convenient and visual reverse transcription-recombinase poly-merase amplification(RT-RPA)combined with lateral flow dipstick(LFD)method for CTV detection.【Methods】Three pairs of primers and a specific probe used for CTV detection were designed accord-ing to the conservative sequence of the coat protein(CP)gene of CTV isolates(NCBI number MH558665.1,MH558666.1,JX266712.1,JQ911664.1 and JQ061137.1)from China.By detecting CTV-infected citrus samples,primers with the best specificity and amplification efficiency were selected to establish the RT-RPA-LFD for CTV detection.The total RNAs were extracted from 100 mg CTV-infect-ed citrus leaf samples using RNAiso Plus and used for CTV detection.The reverse transcription was performed using a C1000 Thermal Cycler in a 20μL reaction mix containing 1μL of Oligo dT Primer,1μL of 10μmol·L^(-1)dNTP Mixture,1μL of RNA template,4μL of PrimeScript Buffer,0.5μL of RNase Inhibitor,and 1μL of PrimeScript RTase.The reaction was carried out for 45 min at 42℃and 5 min at 95℃.RT-RPA-LFD reaction system was optimized with respect to the primer concentration(1,2.5,5,10,20,and 50 nmol·L^(-1)),reaction time(5,10,15,20,25,30,35 and 40 min),and reaction gradient temperature(10,15,20,25,30,35,40,45 and 50℃).For visual detection,LFD strips from the AmplifyRP×RT Discovery Kit were added to the RT-RPA products.The reactions should be al-lowed to incubate for no more than 30 min.The two visual bands of the test and control lines suggested that the tested sample was CTV-positive,and only one band on the control line indicated a negative re-sult.The optimized reaction conditions were determined through the colour density of the test line.The specificity of the established RT-RPA-LFD was evaluated by detecting the samples infected with CTV,citrus yellow vein clearing virus(CYVCV),Citrus tatter leaf virus(CTLV),citrus exocortis viroid(CEVd),citrus psorosis virus(CPV),citrus chlorotic dwarf-associated virus(CCDaV),and the virus-free citrus plants,respectively.To evaluate the detection range of the optimized RT-RPA-LFD,eight CTV genotypes and eleven CTV isolates from different countries were used.A series of 10-fold dilu-tions(2.12×106-2.12×10-1 copies·μL^(-1))of CTV samples were used to test the sensitivity of the RT-RPA-LFD assay,and the sensitivity was compared with the conventional RT-PCR and RT-qPCR.Further-more,the leaves of 67 CTV-suspected different tangor cultivar samples were randomly collected from Chongqing,Sichuan and Guangxi provinces,and used for RT-PCR and RT-RPA-LFD detection.【Re-sults】A RT-RPA-LFD assay for rapid visual detection of CTV was established,with primer pairs RPA-1F(5’-CTTGCTGGCGTCCCTTGTTTCTGTTCTTGTCTT-3’)and RPA-1R(5’-ATTCTGTTTCCTT TCCTAGCCGGGCTTCTTCAC-3’),and RPA-P probe(5’-GGCGAAAAATCTTTTCGTCTACT TG-GTTTTCACTCGCGAAG GCA-3’).It could specifically amplify the target fragment of CTV with a size of 156 bp.The optimal reaction conditions for the determination of RT-RPA-LFD assay were deter-mined as 10μmol·L^(-1)primer concentration,25 min reaction time and 40℃incubation temperature.This method has high specificity to CTV,and no test line was observed when total nucleic acid extracts from CTLV,CYVCV,CEVd,CPV,CCDaV,or healthy citrus plants were tested.This method could also detect different genotypes and origin of CTV.In the sensitivity detection,2.12×10^(1)copies·μL^(-1)was the lowest detection sensitivity of RT-RPA-LFD and RT-qPCR.The limit of detection of RT-PCR was 2.12×103 copies·μL^(-1),indicating that the RT-RPA-LFD method would be 100 times more sensitive than RT-PCR,which was consistent with that of RT-qPCR.Furthermore,the RT-RPA-LFD detection of CTV required shorter detection time(approximately 30 min)than RT-PCR and RT-qPCR.Among 67 citrus samples randomly collected from the field,CTV was detected from 41 samples using RT-RPA-LFD and RT-PCR assay showed the same results.These results suggested that the RT-RPA-LFD method would be suitable for CTV detection in the field.【Conclusion】In this study,a visual RT-RPA-LFD method for CTV detection was developed and the optimal reaction conditions for the RT-RPA-LFD assay were de-termined.The new RT-RPA-LFD method would be more effective and sensitive for the precise quantifi-cation of CTV than RT-PCR.It could be applied to on-site rapid detection for the plant protection and quarantine station.