目的 建立并優(yōu)化水蛭ISSR-PCR反應體系及擴增程序，為水蛭進行遺傳多樣性研究提供依據(jù)。方法 使用引物ISSR825，采用正交優(yōu)化方法對影響PCR反應的Mg²⁺、dNTP、引物、Taq DNA聚合酶、模板DNA進行了體系優(yōu)化，同時對引物的最佳退火溫度進行了選擇。結(jié)果 在25 μL總體積中，其中包括10×PCR 緩沖液 2.5 μL、Mg²⁺ 2.0 mmol/L、dNTP 0.25 mmol/L、引物0.8 μmol/L、Taq DNA聚合酶2.5 U、模板DNA 2.0 ng/μL，最佳退火溫度為49.7 ℃。結(jié)論 所建立的最佳ISSR-PCR反應體系穩(wěn)定可靠，可用于水蛭遺傳多樣性評價、不同種源鑒定及親緣關(guān)系分析。

Abstract: Objective To establish and optimize the ISSR-PCR reaction system and amplification procedure for Hirudo nipponica and to provide the basis for its genetic diversity research. Methods Using Primer ISSR825, the orthogonal test design was adopted to optimize the ISSR-PCR amplification system on H. nipponica in five factors (Mg²⁺, dNTP, primer, Taq DNA polymerase, and template DNA) at four levels, and the suitable anneal temperature of the primer was selected. Results The suitable ISSR-PCR system (25 μL reaction volume) in H. nipponica included 10 × PCR buffer (2.5 μL), Mg²⁺ (2.0 mmol/L), dNTP (0.25 mmol/L), primer (0.8 μmol/L), Taq DNA polymerase (2.5 U), and template DNA (2.0 ng/μL); The suitable anneal temperature was 49.7 ℃. Conclusion The established and optimized ISSR reaction system is stable and reliable, which could provide the basis for the analysis of genetic diversity, germplasm resources, and genetic relationship of H. nipponica.