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目的 比较连续荧光检测法 (荧光法 )与高效液相色谱 (HPLC)法测定细菌染色体鸟嘌呤、胞嘧啶百分比 (GC % )的稳定性、准确性和适应性。方法 应用双股DNA结合染料 (SYBRGreen1)和能够快速升降温度的连续荧光检测仪 ,测定 9株细菌染色体的变性温度℃值 ,并根据相应的公式算出GC % ;以HPLC法直接检测细菌染色体的GC %。结果 DNA纯度对荧光法测得的GC %结果无显著影响 ,但影响HPLC的测定结果 ,荧光法 3次检测获得的 9株细菌GC %有 4株略高于文献值 ,而HPLC法测得的结果与文献值完全相符 ;用荧光法测得的标准差 ,7/ 9的菌株高于HPLC法。结论 荧光法检测细菌染色体GC %具有简便、快速且不受DNA纯度影响等优点 ,适用于临床微生物的快速诊断。HPLC法在DNA高度纯化的基础上测得的GC %准确、可靠、结果稳定 ,适于细菌分类研究 ,然而限于对标本纯度的要求及操作的复杂性 ,难以在临床推广应用。
OBJECTIVE To compare the stability, accuracy and adaptability of the GC% of bacterial chromosomes with continuous fluorescence detection (FITC) and high performance liquid chromatography (HPLC). Methods The denaturation temperature (℃) of nine bacterial chromosomes was determined by double-stranded DNA-binding dye (SYBRGreen1) and a continuous fluorescence detector capable of rapidly increasing and decreasing the temperature. GC% was calculated according to the corresponding formula. The GC %. Results The purity of DNA had no significant effect on the GC% of the results measured by the fluorescence method, but affected the determination results of HPLC. Four of the nine strains of bacteria obtained by the three fluorescence detection methods were slightly higher than the reference value, while the HPLC method The results are in good agreement with the literature values. The standard deviation measured by fluorescence method was higher for the 7/9 strains than for the HPLC method. Conclusion The fluorescence detection of bacterial chromosome GC% is simple, rapid and not affected by the purity of DNA, which is suitable for the rapid diagnosis of clinical microorganisms. The HPLC method is accurate, reliable and stable with the result of highly purified DNA. It is suitable for the research of bacterial classification. However, GC% is limited to the purity of the sample and the complexity of the operation, which makes it difficult to be applied clinically.