Abstract:

【Objective】Establishing a universal PCR detection system would address the issue of inconsistent amplification systems and reaction conditions of different events, thereby enhancing the efficiency of event detection. 【Method】 In this study, we analyzed and compared the differences in amplification systems and reaction conditions of qualitative and quantitative PCR detection methods for the specificity of transgenic material events. The most widely used PCR systems and conditions parameters were selected as unified parameters to validate the PCR qualitative detection methods for different maize events in the laboratory. 【Result】 General PCR amplification system: Total volume 25.0 μL, 25 mmol/L MgCl₂ solution 1.5 μL, 2.5 mmol/L dNTPs mixed solution 2.0 μL, final concentration of upstream and downstream primer 0.4 μmol/L, Taq DNA polymerase 0.025 U/μL, 25 mg/L DNA template 2.0 μL, LOD 0.1%. Reaction conditions: Denaturation at 94℃ for 5 min, 35 cycles of denaturation at 94℃ for 30 s, annealing at 58℃ for 30 s, extension at 72℃ for 30 s, and final extension at 72℃ for 7 min. Universal real-time fluorescent qualitative PCR amplification system: Total volume 20.0 μL, 25 mmol/L MgCl₂ solution 2.0 μL, dNTPs mixed solution（2.5 mmol/L each）1.6 μL, final concentration of upstream and downstream primers and probe 0.4 μmol/L, Taq DNA polymerase 0.04 U/μL, 25 mg/L DNA template 2.5 μL, LOD 0.1%. Reaction conditions: denaturation at 95℃ for 5 min; 95℃ denaturation 15 s, 60℃ annealing extension 60 s, 40 cycles.【Conclusion】This system and its associated amplification system and reaction conditions can be used to detect different event materials.

Key words: event, polymerase chain reaction（PCR）, amplification system, reaction conditions, Zea mays L.