荧光定量PCR评价铜绿假单胞菌感染的实验研究

目的探讨FQ-PCR定量检测铜绿假单胞菌oprI基因的方法在快速诊断铜绿假单胞菌败血症，及快速判定抗生素体内疗效中的作用。方法（1）制备不同浓度的标准菌株并行药敏实验，找出敏感药物（丁胺卡那霉素）及非敏感药（苯唑西林钠）。（2）SD大鼠120只随机分为5组，分别取不同浓度的菌液尾静脉注入，于实验开始0h、12h、24h、48h各组分别麻醉6只大鼠，采血作血培养、FQ-PCR。（3）SD大鼠72只随机分为治疗组、处理组和对照组，均给予1×10^9CFu／ml尾静脉注入。随后治疗组立即给予敏感抗生素；处理组立即给予非敏感抗生素；对照组同时给予等量生理盐水，均于尾静脉注入。各组于首次注射后0h、12h、24h、48h分别麻醉6只大鼠，采血作血培养、FQ-PCR。结果（1）1×10^9CFu／ml、1×10^8CFU／ml组各时间段血培养均为阳性，FQ-PCR检测，均为阳性。（2）1×10^7CFU／ml、1×10^6CFU／ml组0h、12h血培养均为阳性，1×10^7CFU／ml组24h血培养为阳性，48h为阴性，而1×10^6CFU／ml组24h、48h血培养均为阴性。FQ-PCR检测1×10^7CFU／ml组0h、12h、24h为阳性，48h为阴性。1×10^6CFU／ml组0h、12h、为阳性24h、48h为阴性。（3）1×10^5CFU／ml组各时间段血培养均为阴性，FQ-PCR检测均为阴性。（4）处理后对照组、处理组各时间段血培养均为阳性，而处理后治疗组各时间段血培养均为阴性。FQ-PCR检测三组各时段均为阳性。结论（1）FQ-PCR诊断铜绿假单胞菌败血症与传统细菌培养比较其特异性吻合率达100％，虽灵敏度并未增加，但诊断时间明显缩短。（2）有效抗生素治疗后，运用FQ-PCR诊断灵敏度明显高于细菌培养。（3）利用FQ-PCR检测铜绿假单胞菌oprI基因的拷贝数的变化可能有助于抗生素体内敏感性的判断。

The experimental research of the evaluation on Pseudomonas aeruginosa infection with FQ-PCR

Objective To probe the oprⅠ gene in rat model with Pseudomonas aeruginosa septicemia by FQ-PCR,and compare the sensitivity and specificity between FQ-PCR and traditional germiculture,and check the change of oprI gene before and after the antibiotic therapy as to rapidly judge its sensitivity.Methods The standard Pseudomonas aeruginosa with five different concentration were prepared,the drug-sensitive test wbre used to find lhe sensitive antibiotics.120 SD rats were random divided into five groups,five different concentrations of Pseudomonas aeruginosa were injecked into the rats with the same volume.Six rats of each group were picked up for taking blood for culture at the time points of Oh,12h,24h,and 48h after narcotization.Finally,the oprⅠ gene of each blood samples were checked with FQ- PCR.72 rats were random divided into three groups,therapeutic group,treated group and control group.Pseudomonas aeruginosa with the concentration of 1×109 CFU/ml were injected into those rats.Sensitive antibiotics,insensitive antibiotics and 0.9% NaCl were given to the therapeutic,treated and control group rats respectively.Six rats of each group were picked up for taking blood for culture at the time point of Oh,12h,24h,and 48h after narcotized.Finally,the oprⅠ gent of each blood sample were checked with FQ-PCR.Results The blood culture were positive in each period of the concentrations 1×109 CFU/ml and 1×108 CFU/ml.Results of FQ-PCR showed that the copy number decreased with time going,all of which were positive.The blood culture were positive at the time points of Oh and 12h with the concentrations of 1×107 CFU/ml and 1×106 CFU/ml,were positive with concentration of 107 CFU/ml at the time point of 24h,but negative with concentration of 107 CFU/m at the time point of 48h,and negative with the concentration of 1×106 CFU/ml at the time points of 24h and 48h.The blood culture were negative in each period of the concentration of 1×105 CFU/ml,and the results of FQ-PCR were negative.The blood culture were positive in each period of both treated and control group,but negative in each period of therhpeutic group,all the results of FQ-PCR were positive.Conclusion The coincidence rate between the method of FQ-PCR and trgditional germicuhure were 100%.Though the sensitivity of FQ-PCR was not increased,the time needed by diagnosis was shorter After treated with effective antibiotic,fhe sensitivity of FQ-PER to diagnosis Pseudomonas aeruginosa septicemia was higher than that of traditional germicuhure,and the experiment time was shorter.Detected the changes of the oprⅠ gene copies number may be helpful to estimate the sensitivity of antibiotic.