多重PCR检测角膜致病真菌体系优化的探索

Application of a stable PCR system in diagnosis of mycotic keratitis

目的探索多重PCR体系中各成分的最适组合,以便在真菌性角膜炎的诊断中获得准确、可靠的结果。方法对参与PCR基因扩增体系的成分在一定范围内设置不同的浓度或参数组,进行单因子或复因子检测,观测各因子在不同条件下对扩增结果的影响。结果多重PCR体系中各组分及退火温度等都会对反应产生一定的影响。优化后的反应体系体积为30μl:20×PCR Buffer 1.5μl,MgC l22 mmol/L,dNTP 200μmol/L,引物各10 pmol,Taq DNA聚合酶2.0μl,DNA模板2μl;PCR反应参数:预变性94℃300 s,变性94℃60 s,退火55℃60 s,延伸72℃45 s,35个循环,终延伸300 s。结论多重PCR扩增体系通过对各种成分、因子的优化,可以达到特异性高且稳定可靠的目的。

Objective Many factors can influent the sensitivity and specificity of the muhi-PCR amplification system,so it is necessary to explore a suitable condition in multi-PCR for obtaining a proper result in diagnosis of mycotic keratitis. This paper was to optimize the multi-PCR test of mycotic keratitis. Methods A series concentrations of Mg^2＋, primer, Taq DNA polymerase and dNTP were added to the muhi-PCR amplification system. Other ingredient was fixed when adjusted one ingredient. A series annealing temperatures were tested and chosen to optimize the conditions for amplification. The influence of various factors on amplification outcome was observed by gelose gel electrophoresis. Results Different composition of certain concentration played different role in muhi-PCR amplification system. Optimized system had a total volume of 30 μl containing 20 × PCR buffer 1.5 μl,MgCl2 2mmol/L, dNTP 200 μmol/L, primer 10pmol, Taq DNA polymerase 2. 0 μl, DNA template 2 μ1. The reaction was performed as hot started PCR with 300 s initial denaturation at 94℃. The PCR program comprised 35 cycles （60 s at 94℃ ,60 s at 55℃ ,45 s at 72℃ ）. A final elongation of 300 s at 72℃ followed the last cycle. Conclusion Optimization of the multi-PCR can be a highly sensitivity,specificity and stability system.