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.
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