Studies On Molecular Methods For Detection Of Southern Rice Black-Streaked Dwarf Virus

Southern rice black-streaked dwarf virus (SRBSDV) is a suggested new species in the genus Fijivirus group2within the family Reoviridae. The virus is mainly transmitted by white-backed planthopper (WBPH, Sogatella furcifera) to rice and corn respectively resulted in southern rice black-streaked dwarf disease and maize rough dwarf disease in a persistent manner, which could not be transmitted to offspring of WBPH through ovary. The diagnosis of suspected diseased plants and carrier rate of WBPH were the significant factors for disease outbreak forecast. At present, the detection methods for SRBSDV mainly rely on biological, immunological, and molecular biology methods. But these methods existed some of disadvantages, time-consuming, complex operation and expensive etc, which were hard to achieve the ambition of rapid, accurate, low-cost detection for SRBSDV, and which were can’t realize the quantitation of viral RNA copy numbers. In addition, rice plants infected by SRBSDV show typical symptoms similar to rice black streaked dwarf disease. The two viruses also share other similarities such as in genome sequence, virion morphology, serology, insect vector and host ranges. It is hard to distinguish between them by traditional test methods. It is essential to develop detection metods to meet the different places and requirements.The reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for SRBSDV detection was established to realize the rapid, inexpensive, specific and simple demand. Based on the sequences of the highly conserved regions of SRBSDV S9, that were dissimilar to those of RBSDV, four oligonucleotide primers (F3, B3, FIP, BIP) for use in RT-LAMP were designed. We optimized the experimental condition of RT-LAMP from different concentration of primer and MgSO4and different reaction time and temperature. The results suggested that the ratio of primer concentration was1:8(0.2μM of F3/B3,1.6μM of FIP/BIP), the concentration of MgSO4was8mM.60-62℃constant temperature60min was appropriate reaction conditions. The RT-LAMP assay was ten times more sensitive than the RT-PCR method for SRBSDV detection. The RT-LAMP demonstrated a high degree of specificity for SRBSDV, which eliminated the interference of RBSDV and appeared to be suitable for a rapid detection of SRBSDV in field samples of host and vectors.Real time RT-PCR method was established to realize the quantitation of viral RNA copy numbers.We designed two specific oligonucleotide primers (SRBSDV-S9-F and SRBSDV-S9-R) for real time RT-PCR according to the sequences of the highly conserved regions of SRBSDV S9. We got the standard RNA through preparing restructured plasmid pGEM-T easy-S9and vitro transcription and purification. The best experimental conditions (300nM primers,60.0℃annealing-extension) were determined by optimizing the concentration of primers and different annealing-extensions temperature. Viral RNA transcripts were used in10-fold serial dilutions to generate the standard curve, then detecting the melting temperature of amplification products to get the melting curve. The R Square (R2) of standard curve is0.996, and amplification efficiency reach to100.2%. The melting curve showed a single sharp peak, which explained a single amplification product and good specificity. We found that the real time RT-PCR method showed high sensitivity through the sensitivity evaluation, which was100times more sensitive than the RT-PCR method for SRBSDV detection. It can be used for the precisely test of viral copy numbers., which provide reliable basis for the studies of pathogenic mechanism of SRBSDV and molecular biology research.In the study, the specificity, sensitivity and reaction time of RT-LAMP, RT-PCR, Real time RT-PCR and DIBA for SRBSDV detection were compared with each other in order to evaluate the scope of application of each detection methods. The results suggested that RT-LAMP method is suitable to rapidly and accurately detect SRBSDV of plants and insects especially for grassroots departments, real time RT-PCR achieve to precisely quantify the copy numbers of SRBSDV RNA, DIBA were used to detect the virus carried rate WBPH and SBPH, which provide a method for vaccinating against the virus of indoor experiment and detecting the rate of virus carriers.