Establishment Of Reverse Transcription Loop-mediated Isothermal Amplification For Detecting Citrus Tatter Leaf Virus And Its Prokaryotic Expression Of Coat Protein Gene

. Citrus tatter leaf disease caused by Citrus tatter leaf virus (CTLV) is of somewhat economical importance in quite a few countries. CTLV mainly infects Poncirus trifoliata or its hybrids, which are widely used as rootstocks worldwide. The distribution of CTLV is somehow widespread in China due to the wide usage of CTLV-susceptible rootstocks along with the rapid development of citrus industry. Since the symptoms of CTLV usually are hidden within a few years after its infection, thus, it is of somewhat potential threat to the citrus industry. Planting virus-free citrus nursery trees has been most effectively preventing its damage, therefore, it is very important to establish a rapid, reliable and simple method for the detection of CTLV. In this study, a sensitive and accurate reverse transcription loop-mediated isothermal amplification (RT-LAMP) detection system was established. Meanwhile, in order to prepare the specific antibody against CTLV, coat protein of CTLV was cloned and then expressed via prokaryotic expression system. Finally, a colloidal gold immuno-chromatographic strip for the detection of CTLV was preliminarily addressed. The results are summarized as follows:1. RT-LAMP for CTLV detectionA specific and sensitive RT-LAMP system for the detection of CTLV was established with one set of six primers. This system was established through optimizing several factors such as the concentrations of Mg2+, primers, dNTPs and betaine, the reaction time and temperature. The optimized reaction system of a total volume of25μL RT-LAMP was settled down, including1μL of RNA template,2U of Avian myeloblastosis virus (AMV) reverse transcriptase,8U of Bst DNA polymerase,6U of RNase inhibitor, primers F3/B3at0.5μmol/L, primers FEP/BIP at1μmol/L, Loop F/B at0.2μmol/L, MgSO4at6mmol/L, dNTPs at1.2mmol/L, betaine at0.6mmol/L,5μL Reaction Buffer [containing100mmol/L Tris-HCl (pH8.8),50mmol/L KCl,50mmol/L (NH4)2SO4,0.5%Triton X-100]. The optimized temperature is at63℃and the reaction time is for1h. The specificity test of RT-LAMP suggested that there was no cross reaction with Citrus tristeza virus(CTV), Satsuma dwarf virus (SDV), Candidatus Liberibacter asiaticus, Xanthomonas axonopodis pv.citri(Xac), Citrus exocortis viroid (CEVd), Citrus viroid mixtures{contained Citrus bent leaf viroid (CBLVd), Hop stunt viroid (HSVd), Citrus viroid-Ⅲ (CVd-Ⅲ), Citrus viroid-Ⅳ (CVd-IV), Citrus viroid-V (CVd-V), Citrus viroid-OS (CVd-OS) and Vd-I-LSS (Citrus viroid I-low sequence similarity)}, water and the extract of healthy leaf. The high specificity of RT-LAMP was confirmed using CTLV as the positive control.The diagnosis sensitivity of RT-LAMP was compared with one step RT-PCR and quantitative real-time RT-PCR assays. The results showed the detection limit of RT-LAMP and quantitative real-time RT-PCR could reach1.22×10-2ng/μL, whereas of one step RT-PCR reached1.22ng/μL. Therefore, the RT-LAMP was at least100times more sensitive than that of the one-step RT-PCR.2. Clone and prokaryotic expression of coat protein gene of CTLVThe sample infected by CTLV was amplified by RT-PCR with specific primers. RT-PCR product was then purified and inserted into pMD19-T vector. The sequencing result of vector demonstrated that the gene cloned showed100%homology with the CP sequence of CTLV reported on NCBI (GenBank Number, FJ223212).The CP was amplified by RT-PCR with a new primer pairs (PrimerF1/R1). The recombinant plasmid pET-28a-CP was then constructed after CP was inserted into a prokaryotic expression vector pET28a(+). The pET-28a-CP was used to transform Escherichia coli BL21(DE3) and then induced by IPTG About30kD protein was expressed, which was highly homologous with the CP of CTLV.After the pET-28a-CP expressed, the precipitate and supernatant were collected, and then crushed by ultrasonic sterilization. The majority of expressed protein was obtained in precipitate, suggesting that the expressed protein existed in the form of inclusion body. The expressed protein diluted by5000times still showed a specific positive reaction with the antibody against ASGV by Enzyme-linked immuno sorbent assay(ELlSA). The CP was submitted to the Biotechnology Research Institute of Zhejiang University for the preparation of CTLV antibody.3. Preliminary establishment of colloidal gold immuno-chromatographic strip for CTLV detectionA series of colloidal gold with different diameters had been prepared with a series of volume of sodium citrate, the results showed that the diameter of optimal colloidal gold used for this study was20.3nm, which was preprepared by adding0.9mL of2%sodium citrate into100mL colloidal gold solution. The system of colloidal gold immuno-chromatography was preliminarily addressed by setting up the optimal parameters such as pH8.2of the conjugation, O.Olmg antibody per100μL colloidal gold, and0.2mg/mL of the capture antibody. In this study, although the control point could provide a stable, clear result when testing samples were approached, the colloidal gold test strip assembled preliminarily is still in need to improve.