Establishment of a Rapid One-step Multiplex RT-PCR Detection Method for Six Rice Viruses
HUANG Jing1,2, WANG Chen-Yi2, LAN Bin-Yuan2, Ding Xin-Lun2, ZHANG Shuai2, WU Jian-Guo2,*
1 College of Food and Biological Engineering, Zhangzhou Institute of Technology, Zhangzhou 363000, China; 2 Vector-borne Virus Research Center, Fujian Agriculture and Forestry University, Fuzhou 350002, China
Abstract:The occurrence of viral diseases of rice (Oryza sativa) in agricultural production is mostly caused by multiple viruses coinfection. When rice is infected with different viruses, different disease characteristics and complex symptoms or disease-like symptoms caused by multiple viruses are often difficult to diagnose only by virological symptoms. Therefore, according to the current agricultural production of China's 6 common rice viruses, namely, Rice stripe virus (RSV), Rice grassy stunt virus (RGSV), Rice dwarf virus (RDV), Rice ragged stunt virus (RRSV), Rice black-streaked dwarf virus (RBSDV), Rice ragged stunt virus (RRSV) and Southern rice black-streaked dwarf virus (SRBSDV), specific primers were designed according to the coat protein (CP) gene sequence of each virus, respectively. A rapid and simultaneous one-step multiplex reverse transcription PCR (RT-PCR) method was developed to detect the 6 viruses on rice. The results showed that the primer (10 μmol/L) final volume, RSV, RDV, RGSV, RRSV, RBSDV and SRBSDV were 0.6, 0.4, 0.3, 0.4 0.6 and 0.2 μL, respectively. The dosage of OneStep RT/Taq Mix, 5×reaction buffer and the sample RNA of rice virus were determined to be 0.75 μL, 4 μL and 1 μg, respectively. Finally, add DEPC H2O to 20 μL. Multiple RT-PCR parameters were set as RNA reverse transcription at 50 ℃ for 30 min; Pre-denaturation step at 94 ℃ for 2 min; 35 cycle denaturation step at 94 ℃, 30 s, annealed at 58 ℃ for 30 s, extended at 72 ℃ for 1 min; With a final extension at 72 ℃ for 10 min. The research results indicated that the system could be efficient and accurate in distinguishing the 6 rice viruses, greatly improved the efficiency of detection. This method can be widely used in laboratory-based accurate detection, field-based rice virus disease diagnosis and vector detection, and also provides technical support and reference basis for the research and development of related products.
[1] 沈建国, 高芳銮, 蔡伟, 等. 2016. 进境大豆种子上菜豆荚斑驳病毒和大豆花叶病毒的多重RT-PCR检测[J]. 中国农业科学, 49(4): 667-676. (Shen J G, Gao F L, Cai W, et al.2016. Multiplex RT-PCR for simultaneous detection of Bean pod mottle virus and Soybean mosaic virus in imported soybean seeds[J]. Scientia Agricultura Sinica, 49(4): 667-676.) [2] 郑轩. 2010. 五种烟草病毒的多重RT-PCR检测技术研究[D]. 硕士学位论文, 西北农林科技大学, 导师: 吴云锋, 成巨龙, pp. 10-30. (Zheng X.2010. Detection of five tobacco viruses with multiplex RT-PCR[D]. Thesis for M.S, Northwest Agriculture and Forestry University, Supervisor: Wu Y F, Cheng J L, pp. 10-30.) [3] 钟泽澄, 王进, 张师音. 2020. 多重PCR技术研究进展[J]. 生物工程学报, 36(2): 171-179. (Zhong Z C, Wang J, Zhang S Y.2020. Advances in multiple PCR technology studies[J]. Chinese Journal of Biotechnology, 36(2): 171-179.) [4] Chen Z, Liu J, Zeng M, et al.2012. Dot immunobinding assay method with chlorophyll removal for the detection of southern Rice black-streaked dwarf virus[J]. Molecules, 17(6): 6886-6900. [5] Cho S Y, Jeong R D, Yoon Y N, et al.2013. One-step multiplex reverse transcription-polymerase chain reaction for the simultaneous detection of three rice viruses[J]. Journal of Virological Methods, 193(2): 674-678. [6] Hibino H.1996. Biology and eprdemiology of rice viruses[J]. Annual Review of Phytopathology, 34(1): 249-274. [7] Le D T, Netsu O, Uehara-Ichiki T, et al.2010. Molecular detection of nine rice viruses by a reverse-transcription loop-mediated isothermal amplification assay[J]. Journal of Virological Methods, 170: 90-93. [8] Matsukura K, Towata T, Sakai J, et al.2013. Dynamics of southern Rice black-streaked dwarf virus in rice and implication for virus acquisition[J]. Phytopathology, 103(5): 509-512. [9] Parida M, Sannarangaiah S, Dash P K, et al.2008. Loop mediated isothermal amplification (LAMP): A new generation of innovative gene amplification technique; perspectives in clinical diagnosis of infectious diseases[J]. Reviews in Medical Virology, 18(6): 407-421. [10] Uehara-Ichiki T, Shiba T, Matsukura K, et al.2013a. Detection and diagnosis of rice-infecting viruses[J]. Frontiers in Microbiology, 4: 289. [11] Uehara-Ichiki T, Shiba T, Ueno T, et al.2013b. Evaluation of the DAS-ELISA as a detection method for Rice stripe virus from its vector insect, small brown planthopper, Laodelphax striatellus[J]. Japanese Journal of Applied Entomology and Zoology, 57(2): 113-116. [12] Wang H, Xu D, Pu L, et al.2014. Southern Rice black-streaked dwarf virus alters insect vectors' host orientation preferences to enhance spread and increase rice ragged stunt virus co-infection[J]. Phytopathology, 104(2): 196-201. [13] Wang Q, Yang J, Zhou G H, et al.2010. The complete genome sequence of two isolates of southern Rice black‐streaked dwarf virus, a new member of the genus Fijivirus[J]. Journal of Phytopathology, 158(11-12): 733-737. [14] Wu W Q, Guo X G, Zhang H M, et al.2013. Simultaneous detection and survey of three rice viruses in China[J]. Plant Disease, 97(9): 1181-1186. [15] Yang X, Lv K L, Wang M Q, et al.2017. Investigation of viruses infecting rice in southern China using a multiplex RT-PCR assay[J]. Crop Protection, 91: 8-12. [16] Zhang P, Mar T T, Liu W, et al.2013. Simultaneous detection and differentiation of Rice black streaked dwarf virus (RBSDV) and Southern rice black streaked dwarf virus (SRBSDV) by duplex real time RT-PCR[J]. Virology Journal, 10: 24. [17] Zhang X, Wang X, Zhou G.2008. A one-step real time RT-PCR assay for quantifying Rice stripe virus in rice and in the small brown planthopper (Laodelphax striatellus Fallen)[J]. Journal of Virological Methods, 151: 181-187.