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目的建立高效液相色谱技术(HPLC)检测(13)βD葡聚糖的方法,并与巢式聚合酶链反应(PCR)方法进行比较,评价其在实验大鼠侵袭性肺曲霉病(IPA)早期诊断中的意义。方法将烟曲霉孢子注入实验大鼠左肺以制作IPA模型;采集各组实验大鼠血样及脏器标本,进行巢式PCR、HPLC及培养方法的检测。结果HPLC检测(13)βD葡聚糖最低检测限度为1～2pg/ml,临界值为15pg/ml。模型组检测值和阳性率均较对照组高,差异有统计学意义(P<0.01);随感染时间延长检测值和阳性率逐渐上升,各组差异有统计学意义(P<0.01);感染死亡大鼠血液标本的检测值与死亡时间呈负相关关系,其相关系数r为-0.949(P<0.01)。1周内HPLC方法敏感度(77.8%)和特异度(91.7%)均较巢式PCR高,但差异无统计学意义(P>0.05),两者敏感度均高于血培养,差异均有统计学意义(P<0.01)。结论HPLC检测大鼠血中(1～3)βD葡聚糖浓度,在早期预测大鼠肺曲霉感染的发生、发展及预后判断方面明显优于传统的血培养,也要优于巢式PCR方法。
OBJECTIVE: To establish a HPLC method for the determination of (13) β-D-glucan and to compare it with nested polymerase chain reaction (PCR) The significance of early diagnosis. Methods Aspergillus fumigatus spores were injected into the left lung of experimental rats to make IPA model. Blood samples and viscera samples from each group were collected and tested by nested PCR, HPLC and culture methods. Results The minimum detection limit of (13) βD dextran by HPLC was 1 ~ 2 pg / ml and the critical value was 15 pg / ml. The detection value and the positive rate in the model group were higher than those in the control group (P <0.01). The detection value and the positive rate increased with the extension of the infection time, the difference was statistically significant (P <0.01) There was a negative correlation between the blood samples and the time of death in the dead rats, and the correlation coefficient r was -0.949 (P <0.01). Compared with nested PCR, the sensitivities (77.8%) and specificity (91.7%) of HPLC method were higher in 1 week than those in nested PCR, but the difference was not statistically significant (P> 0.05) Statistical significance (P <0.01). Conclusion The detection of (1 ~ 3) β D-glucan in rat blood by HPLC was superior to traditional blood culture in predicting the occurrence, development and prognosis of pulmonary aspergillosis in rats in the early stage, and better than the nested PCR .