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

Abstract：【Objective】Tristeza caused by citrus tristeza virus (CTV) is one of the most destructive citrus 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 cultivars. In recent years, stunted, severe stem pitting and reduced fruit quality were observed in Newhall navel orange and some tangor cultivars, causing severe economic losses in major citrus-growing provinces of China, especially in Hunan, Jiangxi, Yunnan, Sichuan provinces. Prompt and accurate CTV detection in the nursery and field samples is necessary to control CTV. To date, serological techniques, reverse 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 polymerase 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 according to the conservative sequence of the coat protein (CP) gene of CTV isolates (NCBI numberMH558665.1, MH558666.1, JX266712.1, JQ911664.1 and JQ061137.1) from China. By detecting CTVinfected 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-infected citrus leaf samples using RNAiso Plus and used for CTV detection. The reverse transcription was performed using a C1000 Thermal Cycler in a 20 μL reaction mix containing 1 μL of Oligo dT Primer, 1 μL of 10 μmol·L^-1 dNTP Mixture, 1 μL of RNA template, 4 μL of PrimeScript Buffer, 0.5 μL of RNase Inhibitor, and 1 μL 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 allowed 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 result. 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 virusfree 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 dilutions (2.12×106 -2.12×10-1 copies·μL-1 ) of CTV samples were used to test the sensitivity of the RT-RPALFD assay, and the sensitivity was compared with the conventional RT- PCR and RT- qPCR. Furthermore, 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.【Results】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 TGGTTTTCACTCGCGAAG 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 determined as 10 μmol · 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, 2.12×101 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 determined. The new RT-RPA-LFD method would be more effective and sensitive for the precise quantification of CTV than RT-PCR. It could be applied to on-site rapid detection for the plant protection and quarantine station.