目的 建立并优化土鳖虫基因组DNA的RAMP-PCR反应体系及扩增程序，为土鳖虫遗传多样性的研究提供依据。方法 以雌性土鳖虫为材料，常规酚/氯仿提取基因组DNA。使用RAMP引物（ISSR807＋RAPD A₅），采用正交优化方法对影响PCR反应的Mg²⁺、dNTPs、引物、DNA聚合酶、模板进行体系优化，同时选择最佳的引物退火温度。结果 最适引物（ISSR807＋RAPD A₅）组合进行土鳖虫的RAMP-PCR分析的反应体系为总体积25 μL，包括10×缓冲液2.5 μL、Mg²⁺ 1.00 mmol/L、dNTPs 2.00 mmol/L、引物0.50 μmol/L、TaqDNA聚合酶1.00 U、DNA模板1.50 ng/μL，最佳退火温度为46.8 ℃。结论 本实验建立的最佳RAMP-PCR反应体系稳定、重复性好，可用于土鳖虫种质资源鉴定和遗传多样性研究。

Objective To provide the basis for the genetic diversity research of Eupolyphaga sinensis by establishing and optimizing the random amplified microsatellite polymorphism RAMP-PCR reaction system and amplification procedure for genomic DNA. Methods Phenol chloroform extraction of genomic DNA was performed on female E. sinensis. Based on RAMP Primer (ISSR807 + RAPD A₅), the orthogonal test was adopted to optimize the RAMP-PCR amplification system on E. sinensis in five factors (Mg²⁺, dNTPs, primer, Taq DNA polymerase, and template DNA) at four levels, and the suitable anneal temperature of the primer was selected. Results The optimal Primer (ISSR807＋RAPD A₅) for RAMP-PCR system (25 μL reaction volume) of E. sinensis was: 10 × PCR buffer (2.5 μL), Mg²⁺ (1.0 mmol/L), dNTPs (2.0 mmol/L), primer (0.5 μmol/L), Taq DNA polymerase (1 U), and template DNA (1.5 ng/μL); The optimized anneal temperature was 46.8 ℃. Conclusion The established and optimized RAMP-PCR reaction system is stable and repeatable, which could provide the basis for the analysis of germplasm resources and genetic diversity of E. sinensis.