VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY -------oOo------- UNDERGRADUATE THESIS TITLE: DEVELOPMENT AND APPLICATION OF A DOT- ELISA 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 DOT- ELISA 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. 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. ii LIST OF FIGURE .v LIST OF TABLE. vi LIST OF ABBREVIATIONS.
Harmful rice viruses. Southern rice black-streaked dwarf virus. The origin of SRBSDV. Pathological characteristics of SRBSDD.
Biological characteristics of SRBSDV. Diagnosis and detection of SRBSDV. Enzyme-linked immunosorbent assay. SRBSDV in Vietnam.
A history of SRBSDD in Vietnam. SRBSDV researches in Vietnam. MATERIALS AND METHODS. Materials, chemicals and equipment.
RNA extraction and quantification. Protein extraction and quantification. Evaluation of anti-SRBSDV antibodies. Optimization of chemical components for SRBSDV test.
Optimization of dot-ELISA assay for SRBSDV diagnosis. Experimentation of optimized dot-ELISA for SRBSDV diagnosis. Assessment of shelf life of SRBSDV diagnostic kit. Assessment of sensitivity and specificity of SRBSDV diagnostic kit.
RESULTS AND DISCUSSIONS. Assessment of anti-SRBSDV antibodies. Assessment of anti-SRBSDV antisera. Assessment of anti-P10 antibody.
Optimization dot-ELISA assay for SRBSDV diagnosis. Optimization of dot-ELISA assay. Diagnosis of SRBSDV with optimized dot-ELISA assay. Development of dot-ELISA kit for SRBSDV diagnosis.
Production of the dot-ELISA kit. Specificity and sensitivity of SRBSDV dot-ELISA kit. Assessment of the shelf life of SRBSDV dot-ELISA kit. CONCLUSIONS AND SUGGESTIONS.57 iv LIST OF FIGURE Figure 1.
Symptoms of SRBSDV-infected rice…………………………………. Titer of the four anti-SRBSDV antisera. Titer of anti-P10 antibody assessed by dot-ELISA…………………. Specificity of anti-P10 antibody…………………………………….
Effect of primary antibody dilution on SRBSDV dot-ELISA test…. Effect of secondary antibody dilution on SRBSDV dot-ELISA test…37 Figure 3. Effect of different buffers on SRBSDV dot-ELISA test……………. Effect of blocking solution on SRBSDV dot-ELISA test…………….
Effect of NaN3 on SRBSDV dot-ELISA test. Effect of TWEEN-20 on SRBSDV dot-ELISA test…………………. Effect of sampling procedure on SRBSDV dot-ELISA test………. Effect of sample storage condition on SRBSDV dot-ELISA test….
Chlorophyll removal efficiency of methanol treatment……………. Effect of blocking duration on SRBSDV dot-ELISA test…………. Effect of primary antibody incubation duration on SRBSDV dot- ELISA test…………………………………………………………………. Effect of secondary antibody incubation duration on SRBSDV dot- ELISA test………………………………………………………………….
SRBSDV diagnosis with RT-PCR method…………………………. SRBSDV diagnosis with optimized dot-ELISA assay………………49 Figure 3. SRBSDV dot-ELISA kits……………………………………………51 Figure 3. Specificity of dot-ELISA-based SRBSDV diagnostic kit………….
Sensitivity of the SRBSDV dot-ELISA kit…………………………. Effect of preservation period on SRBSDV dot-ELISA test…………54 v LIST OF TABLE Table 2. Oligonucleotide sequences used in the study…………………………. SDS-PAGE 12% gel preparation………………………………………23 Table 3.
Results of SRBSDV diagnosis with optimized dot-ELISA assay……. 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 INTRODUCTION Vietnam is one of the largest rice-producing countries in the world. Thus, rice producing is vital for about 70% of the agricultural population and for the food security of the nation. However, rice production is heavily affected by factors such as the loss of area of cultivation, diseases, climate change.
Virus is one of the most dangerous pathogens and is also the cause of instability in rice production and endangered food security, especially with how climate change helps spreading virus diseases with growing frequency and virulent. Virus diseases when happened often cause detrimental effects, which can occur in-turn and re-emerge after a certain period of time. The annual losses caused by WBPH and virus diseases are approximately billions of Vietnam Dong. Southern rice black-streaked dwarf virus (SRBSDV) was first observed in Guangdong province in 2001 and identified in 2008.
In Vietnam, SRBSDV was observed in 2009 and caused major losses on thousands of hectares in North and Central Vietnam. The disease also caused extensive crop damage in 2010, but soon disappeared, and then reemerged strongly in 2017. The SRBSDV is transmitted by white-backed planthopper (WBPH), and the main mode of transmission is the feeding process rather than spawning. For SRBSDV, it is ineffective to start control the disease when rice plants have already shown the symptoms.
Early detection of the virus content in plant samples (symptomatic/asymptomatic) does not bring much significant difference to disease control either. On the contrary, early detection of viruliferous WBPHs in the field is tremendously valuable to southern rice black-streaked dwarf disease (SRBSDD) control that allows farmers to actively eliminate WBPHs, which minimize the risk of SRBSDD emergence. The current laboratory diagnosis of SRBSDV disease employs reverse transcription polymerase chain reaction (RT-PCR) and immunoassays to detect viral RNA in plant samples. Diagnosis by RT-PCR can achieve results with high 1 accuracy, but comes at a high price, and requires a trained laboratory technician that makes it unable to be applied on a large scale.
Meanwhile, immunoassays also provide reliable results with reasonable price, which fits perfectly with the economic situation in Vietnam. The challenging aspect of developing a SRBSDV diagnostic kit based on antigen-antibody interaction is to be able to produce a specific antibody with enough sensitivity to detect SRBSDV in rice plant and WBPH samples. While SRBSDV quick stick test is not available because of the limit of monoclonal antibody (MAb) technology, SRBSDV enzyme-linked immunosorbent assay (ELISA) using polyclonal antibody (PAb) is more compatible with virus diagnosis in Vietnam, in comparison with RT-PCR test. Essentially, Dot-ELISA is a simple, rapid and scalable procedure for screening a large number of samples at one time.
The overall aim of this study is to successfully develop a specific and sensitive diagnostic kit for SRBSDV at a reasonable price. In order to achieve such goal, several requirements need to be attained, including: Dot-ELISA assay is conducted with optimized protocol so that visible dots have clear, bright color with no background. Anti-SRBSDV antibody is specific and sensitive to SRBSDV. Expression of desired P10 protein can be used as positive control in dot-ELISA assay.
Infected rice plant and viruliferous WBPH samples can be seen after color development and virus-free rice and WBPH samples must not exhibit any signal. Harmful rice viruses Rice-infecting viruses have been persistent and are considered one of the most dangerous threats to rice plants at global scale, especially Vietnam. However, it was not until 1895 that the Japanese scientists began to tackle this problem and for the first time, Rice dwarf virus (RDV) was identified. After that, Rice black- streaked dwarf virus (RBSDV) was identified (Shinkai, 1958) and many other rice- infecting viruses as well like Rice tungro bacciliform virus (RTBV), Rice transitory yellowing virus (RTYV), Rice necrosis mosaic virus (RNMV), Rice ragged stunt virus (RRSV), Rice grassy stunt virus (RGSV), Rice hoja blanca virus (RHBV), etc (Hibino, 1979).
Until now, although there is about 30 rice-infecting viruses has been reported, but only 5 of them are common and causing major damages on rice plants (RGSV, RRSV, RTBV, RBSDV, and RTYV) (Hibino, 1996; Zhou et al. In Vietnam, rice-infecting virus disease has caused damage for crops since 1910 in Northern Vietnam, but it was not until 1963 that there was a report of yellowing on rice and in 1965, virus was identified as the cause of transitory yellowing and vector for transmission was green paddy leafhopper. In 1979, the first rice ragged stunt virus image was taken on electron microscopes and followed by investigative, identifying studies and artificial innoculation of rice virus. The development of science and technology has taken researches on rice viruses in Vietnam to the next step as diagnostic methods are becoming more and more diverse such as antiserological methods and PCR method (Vu Trieu Man et al.
In 2006, the cause of Rice yellow dwarf disease (RYDD) in the Mekong Delta was determined as a combination of three viruses: RGSV, RRSV and Tungro spherical virus. This discovery was also verified by International Rice Research Institute (IRRI) specialists using antisera of these three (Pham Van Du, 2006). In 3 2006, the epidemic of rice yellow dwarf and rice ragged stunt disease broke out across Mekong Delta provinces with an infected rice area of about 500,000 hectares and caused a total loss of about 2,000 billion VND. In 2009, a new virus called SRBSDV caused black-striped dwarf disease in 20 northern and central provinces with a total infected area of more than 42,000 hectares.
All rice viruses found in Vietnam are of economic importance and spread through hoppers. The diagnosis is often difficult because the symptoms can be confused by the use of herbicide or physiological effects. Virus detection on WBPH requires diagnostic techniques such as ELISA or PCR. Southern rice black-streaked dwarf virus 1.
The origin of SRBSDV In 2001, a new rice stunt disease was first observed on rice plants (Oryza sativa) in Guangdong, Southern China.