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VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY -oOo - UNDERGRADUATE THESIS TITLE: DEVELOPMENT AND APPLICATION OF A DOTELISA ASSAY FOR DIAGNOSIS OF SOUTHERN RICE BLACK-STREAKED DWARF DISEASE IN THE FIELD HANOI, 3/2020 VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY -oOo - UNDERGRADUATE THESIS TITLE: DEVELOPMENT AND APPLICATION OF A DOTELISA ASSAY FOR DIAGNOSIS OF SOUTHERN RICE BLACK-STREAKED DWARF DISEASE IN THE FIELD Student : La Duc Duy Class : K61CNSHE Faculty : Biotechnology Supervisors : Nguyen Duy Phuong, PhD Bui Thi Thu Huong, PhD HANOI, 3/2020 COMMITMENT I would like to assure that this thesis was completely conducted by scientific researches by myself under the guidances of Dr Nguyen Duy Phuong Department of Plant Molecular Pathology, part of Agricultural Genetics Institute and Dr Bui Thi Thu Huong - Faculty of Biotechnology, Vietnam National University of Agriculture, I assure you that this is my own research The data and results stated in the thesis are honest and have not been published in any other research project I assure you that the information quoted in the thesis has been clearly indicated with the source, with the quotation in accordance with the regulations I take full responsibility for this assurance Ha Noi, March, 2021 Student La Duc Duy i ACKNOWLEDGEMENTS To complete my graduation thesis, in addition to my hard work, I have also received a lot of dedicated helps and guidances from others First and foremost, I would like to express my sincere gratitude to my advisors Dr Nguyen Duy Phuong and Dr Bui Thi Thu Huong for their continuous support of my study and research, for being patient, enthusiastic, and knowledgable Their guidances have helped me in all the time of research and writing of this thesis I could not have imagined having better advisors and mentors for my study After having been taken under their wings, I can feel that I have grown not only as an inspired student but also as a man My sincere thanks also go to Assoc Prof Ha Viet Cuong from our univiersity, MSc Phung Thi Thu Huong, and other people in molecular pathology department, of Agriculture Genetic Institute for offering me an opportunity to work along side them and leading me on my very first project Last but not the least, I would like to appreciate my family, my friends for supporting me mentally throughout my work ii Contents COMMITMENT i ACKNOWLEDGEMENTS ii LIST OF FIGURE v LIST OF TABLE vi LIST OF ABBREVIATIONS vii INTRODUCTION 1 PART I OVERVIEW 3 1.1 Harmful rice viruses 3 1.2 Southern rice black-streaked dwarf virus 4 1.2.1 The origin of SRBSDV 4 1.2.2 Pathological characteristics of SRBSDD 6 1.2.3 Biological characteristics of SRBSDV 8 1.3 Diagnosis and detection of SRBSDV .10 1.3.1 RT-PCR 10 1.3.2 Enzyme-linked immunosorbent assay .13 1.4 SRBSDV in Vietnam .15 1.4.1 A history of SRBSDD in Vietnam .15 1.4.2 SRBSDV researches in Vietnam .17 PART II MATERIALS AND METHODS 20 2.1 Research subject 20 2.2 Materials, chemicals and equipment .20 2.2.1 Materials .20 2.2.2 Chemicals 21 2.2.3 Equipment 21 iii 2.2 Methods 21 2.2.1 RNA extraction and quantification 21 2.2.2 Protein extraction and quantification .23 2.2.3 Evaluation of anti-SRBSDV antibodies 26 2.2.4 Statistical analysis 26 2.2.5 Optimization of chemical components for SRBSDV test 27 2.2.6 Optimization of dot-ELISA assay for SRBSDV diagnosis .29 2.2.7 Experimentation of optimized dot-ELISA for SRBSDV diagnosis 30 2.2.8 Assessment of shelf life of SRBSDV diagnostic kit 31 2.2.9 Assessment of sensitivity and specificity of SRBSDV diagnostic kit .31 PART III RESULTS AND DISCUSSIONS 32 3.1 Assessment of anti-SRBSDV antibodies .32 3.1.1 Assessment of anti-SRBSDV antisera .32 3.1.2 Assessment of anti-P10 antibody .33 3.2 Optimization dot-ELISA assay for SRBSDV diagnosis 35 3.2.1 Optimization of dot-ELISA assay 35 3.2.2 Diagnosis of SRBSDV with optimized dot-ELISA assay 48 3.3 Development of dot-ELISA kit for SRBSDV diagnosis .51 3.3.1 Production of the dot-ELISA kit .51 3.3.2 Specificity and sensitivity of SRBSDV dot-ELISA kit 52 3.3.3 Assessment of the shelf life of SRBSDV dot-ELISA kit 54 PART IV CONCLUSIONS AND SUGGESTIONS 56 4.1 Conclusion 56 4.2 Suggestion 56 REFERENCES 57 iv LIST OF FIGURE Figure 1.1 Symptoms of SRBSDV-infected rice………………………………… Figure 3.1 Titer of the four anti-SRBSDV antisera 32 Figure 3.2 Titer of anti-P10 antibody assessed by dot-ELISA………………… 34 Figure 3.3 Specificity of anti-P10 antibody…………………………………… 35 Figure 3.4 Effect of primary antibody dilution on SRBSDV dot-ELISA test… 36 Figure 3.5 Effect of secondary antibody dilution on SRBSDV dot-ELISA test…37 Figure 3.6 Effect of different buffers on SRBSDV dot-ELISA test…………… 38 Figure 3.7 Effect of blocking solution on SRBSDV dot-ELISA test…………….39 Figure 3.8 Effect of NaN3 on SRBSDV dot-ELISA test 40 Figure 3.9 Effect of TWEEN-20 on SRBSDV dot-ELISA test………………….42 Figure 3.10 Effect of sampling procedure on SRBSDV dot-ELISA test……… 43 Figure 3.11 Effect of sample storage condition on SRBSDV dot-ELISA test… 44 Figure 3.12 Chlorophyll removal efficiency of methanol treatment…………… 45 Figure 3.13 Effect of blocking duration on SRBSDV dot-ELISA test………… 46 Figure 3.14 Effect of primary antibody incubation duration on SRBSDV dotELISA test………………………………………………………………… 47 Figure 3.15 Effect of secondary antibody incubation duration on SRBSDV dotELISA test………………………………………………………………… 48 Figure 3.16 SRBSDV diagnosis with RT-PCR method………………………….49 Figure 3.17 SRBSDV diagnosis with optimized dot-ELISA assay………………49 Figure 3.18 SRBSDV dot-ELISA kits……………………………………………51 Figure 3.19 Specificity of dot-ELISA-based SRBSDV diagnostic kit………… 53 Figure 3.20 Sensitivity of the SRBSDV dot-ELISA kit………………………….53 Figure 3.21 Effect of preservation period on SRBSDV dot-ELISA test…………54 v LIST OF TABLE Table 2.1 Oligonucleotide sequences used in the study………………………….20 Table 2.2 SDS-PAGE 12% gel preparation………………………………………23 Table 3.1 Results of SRBSDV diagnosis with optimized dot-ELISA assay…… 49 Table 3.2 Components of dot-ELISA kit…………………………………………52 vi LIST OF ABBREVIATIONS AGI Agricultural Genetics Institute ANOVA Analysis of variance APS Ammonium persulfate AP Alkaline phosphatase BCIP 5-bromo-4-chloro-3-indolyl phosphate BPH Brown WBPH BSA Bovine serum albumin cDNA Complementary DNA DAS-ELISA Double antibody sanwich ELISA DEPC Diethyl pyrocarbonate Dot-ELISA Dot - enzyme-linked immunosorbent assay EDTA Ethylenediaminetetraacetic acid ELISA Enzyme-linked immunosorbent assay EtBr Ethidium bromide FDV Fiji disease virus IRRI International rice research institute MAb Monoclonal antibody MARD Ministry of Agriculture and Rural Development MRDV Maize rough dwarf virus NBT Nitro blue tetrazolium PAb Polyclonal antibody PBS Phosphate-buffered saline PPD Plant protection department PPRI Plant Protection Research Institute vii PVDF Polyvinylidene fluoride RT-PCR Reverse transcription polymerase chain reaction RBSDV Rice black-streaked dwarf virus RBSDV-2 Rice black-streaked dwarf virus-2 RDV Rice dwarf virus RGSV Rice grassy stunt virus RHBV Rice hoja blanca virus RNMV Rice necrosis mosaic virus RRSV Rice ragged stunt virus RTYV Rice transitory yellowing virus RTBV Rice tungro bacciliform virus RYDD Rice yellow dwarf disease RYSV Rice yellow stunt virus RT Room temperature SBPH Small brown planthopper SDS – PAGE Sodium dodecyl sulfate–polyacrylamide gel electrophoresis SRBSDD Southern rice black-streaked dwarf disease SRBSDV Southern rice black-streaked dwarf virus SD Standard deviation TAE Tris-acetate-EDTA TBS Tris-buffered saline UV Ultra violet WBPH White-backed planthopper viii rate ranging from 91.6% to 95.5% For suspected rice plant samples (asymptomatic), the detection rate was ranging from 57.1% to 82.1% This result could be explained by the logic that the virus content in some samples were below the detection threshold of dot-ELISA Table 3.1 Results of SRBSDV diagnosis with optimized dot-ELISA assay No Type of sample SRBSDV-positive samples No of samples RTDotPCR1 ELISA2 Ratio of successful diagnosis (%)* Rice plants have been inoculated with SRBSDV for 15 to 20 days (symptomatic) 135 135 129 95.5 Rice plants have been inoculated with SRBSDV for to 10 days (asymptomatic) 28 28 23 82.1 Virus-free rice plants in green house 10 0 100 WBPHs fed on rice plants with symptomatic SRBSDD 27 25 19 77.7 Virus-free WBPHs in green house 50 0 100 Field-collected rice plants with clear SRBSDD symptom 14 12 11 92.9 Suspected field-collected rice plants 54 94.4 RT-PCR assay derived from the national project “Development of southern rice black-streaked dwarf virus diagnostic procedure using molecular biology techniques” (2011-2014) project Optimized Dot-ELISA using purified IgG anti-P10 antibody * The accurate diagnosis rate of SRBSDV diagnosis assay by dot-ELISA method was calculated using the following formula: (the number of samples examined by dot-ELISA that had similar result as those examined by RT-PCR/the total examined samples) x 100% 50 In conclusion, over 318 samples were examined using optimized dot-ELISA assay, which reached the accuracy rate of 93.4% when compared to RT-PCR method There was no false positive across all examined samples 3.3 Development of dot-ELISA kit for SRBSDV diagnosis 3.3.1 Production of the dot-ELISA kit The dot-ELISA kit for SRBSDV diagnosis was packaged under two forms, including Maxi KIT (350 tests), and Mini KIT (70 tests) (Fig 3.18) Components for our kit are presented in table 3.2 The kit is preserved and transported under 4oC condition Figure 3.18 SRBSDV dot-ELISA kits Maxi KIT (350 tests) and Mini KIT (70 tests) 51 Table 3.2 Components of dot-ELISA kit Quantity Components Origin Maxi KIT Mini KIT PVDF membrane (5 x cm) 35 dots x 10 membranes 35 dots x membranes Clever (England) Plastic sample grinding pestle 01 01 Biologix (China) Plastice petri dish 01 01 Biologix (China) Filter paper 10 02 Biologix (China) BSA 2.2 g 0.4 g Biobase (China) NaN3 10% 1.0 mL 0.2 mL Merck (Germany) Skim milk 15 gr 3.0 gr Titan (India) 20 X TBST buffer 50 mL x 50 mL Merck (Germany) 10 X substrate buffer 5.0 mL 1.0 mL Merck (Germany) Anti-P10 antibody 12 µL 2.5 μL Self-produced Anti-IgG antibody 20 µL 3.0 μL Promega (U.S.A) NBT (50 mg/mL) 150 µL 30 μL Promega (U.S.A) BCIP (50 mg/mL) 75 µL 15 μL Promega (U.S.A) P10 (50 ng/µL) 150 µL 30 μL Self-produced Manual book 01 01 Self-produced 3.3.2 Specificity and sensitivity of SRBSDV dot-ELISA kit To determine the accuracy of the kit, rice plant samples with different virus diseases, and WBPH samples were examined The detection result (Fig 3.19) shows that only samples had SRBSDV were positive, while RRSV-, and RGSV- 52 infected rice plants as well as virus-free samples were negative Thus, the SRBSDV dot-ELISA kit has been proved to be specific Figure 3.19 Specificity of dot-ELISA-based SRBSDV diagnostic kit DotELISA-based SRBSDV diagnostic kit was used to examine virus-free rice (HR) and WBPH (HP), viruliferous WBPH (IP-SRBSDV) and SRBSDV- (IRSRBSDV), RGSV- (IR-RGSV), RRSV-infected rice (IR-RRSV) TBS: blank sample The results are shown from three biological replicates (I, II & III) Figure 3.20 Sensitivity of the SRBSDV dot-ELISA kit Samples with SRBSDV were examined by both RT-PCR and dot-ELISA Total RNA samples extracted from SRBSDV-infected rice plants (RT-PCR), and SRBSDV-infected rice extracts (Dot-ELISA) were diluted at concentration of 1:1, 1:5, 1:10, 1:20, 1:50 and 1:100, respectively (+): positive control; (-): negative control; M: kb DNA marker (iNtRoN) The result of sensitivity experiment (Fig 3.20) shows that our kit can detect SRBSDV in sample at a dilution of 1:20 compared to sample at 1:50 dilution of RT-PCR Thus, the kit can detect the presence of SRBSDV in one sample (more 53 than one WBPH individual) at the minimum rate of – 10% (number of viruliferous WBPHs/total WBPHs) In conclusion, dot-ELISA-based SRBSDV diagnostic kit can fulfill the requirements for virus disease diagnosis, and can be applied for local plant protection stations 3.3.3 Assessment of the shelf life of SRBSDV dot-ELISA kit For every diagnostic kit, durability is extremely vital, especially for kits that required low temperature storage The components of our kit were prepared in solution containing NaN3 (0.02%) to improve shelf life The storage condition for both before and during application process was optimized at 2oC – 6oC to match the practical condition in Vietnam Figure 3.21 Effect of preservation period on SRBSDV dot-ELISA test Virusfree rice extracts, SRBSDV-infected rice extracts, and P10 protein were used for diagnostic kit preserved at 4oC for 0, 1, 2, 4, 8, 12, 16, 20, 24 weeks, respectively The data are shown as the mean ± SD from three biological replicates Letters with one asterisk and no asterisk represent SRBSDV-infected rice and purified P10 protein, respectively The values marked with the same letter are significantly different (P < 0.05) 54 To determine the storage shelf life, our kit after packaging was preserved at 4oC for different period of time, respectively Test result (Fig 3.21) shows that the kit can be used normally after stored for six months at 4oC (unused until tested) Based on the result of specificity, sensitivity, and shelf life experiment, our SRBSDV diagnostic kit has met the minimum requirement for a diagnostic kit However, the dot-ELISA kit still need to be further experimented to determine the maximum shelf life for each components, especially for anti-P10 antibody and P10 protein (positive control sample) 55 PART IV CONCLUSIONS AND SUGGESTIONS 4.1 Conclusions Selected the anti-SRBSDV antiserum (anti-P10) with the best titer result (> 1:500,000) Assessed purified anti-P10 antibody titer (> 1:100,000) and specificity (bound specifically to SRBSDV) Succeeded in optimizing of dot-ELISA assay for SRBSDV diagnosis Chemical agents (antibody, buffer, blocking solution, TWEEN-20, NaN3), and sampling procedure used in the assay were assessed; the effect of incubation duration was also studied In experimentation test, dot-ELISA can detect SRBSDV in viruliferous samples with 93.4% accuracy rate compared to standard RT-PCR method Successfully developed SRBSDV dot-ELISA kit based on anti-P10 antibody Our SRBSDV diagnostic kit can specifically detect samples carrying SRBSDV while showed no specific reaction to other plant viruses (RGSV, RRSV) and virus-free samples, and can detect SRBSDV in samples at a dilution of 1:20 (50 µg/ µL) The dot-ELISA kit can be stored for a minimum of six months at 2oC – 6oC 4.2 Suggestions - Develop SRBSDV dot-ELISA kit commercially - Continue to develop specific antibodies for other plant virus, especially rice plant such as RRSV, RGSV, etc to produce early detection kits for plant viruses, which helps to control virus diseases 56 REFERENCES Amero S.A., James T.C & Elgin S.C (1988) Production of antibodies using protein in gel bands Methods Mol Biol 3: 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Appendix Assessment of IgG antibody’s purity by SDS-PAGE Appendix Protein sample seperated on 12% polyacrylamide gel Lane M: protein ladder (Intron); lane 1: purified IgG antibody 64
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