目的:确定一个种特异性的实时聚合酶链反应(PCR)法检测绿脓杆菌(PA),一个可能提供给其他细菌病原体通用目标的用于PA的次一级目标DNA,并验证这种用于诊断测试的检测方法。方法:利用已知PA的角膜炎株建立对ecfXPA基因和16SrRNA基因的PCR检测。两个实验的结果参数是"检测限"(LOD),扩增效率(AE)和聚丙烯酰胺凝胶电泳(PAGE)扩增产物分析。两种检测方法通过对20例PADNA阳性的真阳性临床标本和20例不含有PADNA的真阴性样品的检测进行了验证。描述性统计和PAGE分析用作为结果参数。结果:通过ecfX检测的AE为96.6%,而LOD为每微升33.6目标DNA拷贝。16SrRNA检测的AE为103.4%,而LOD为每微升8.12拷贝。ecfX和16SrRNA检测的敏感性,特异性,阳性预测值,阴性预测值,效率分别为(75%,95%,94%,79%和85%),及(70%,100%,100%,77%和85%)。这两种PCR方法经过了验证,通过了PAGE分析的DNA图谱确认。结论:这里描述的PCR方法可能是对PA角膜炎标准诊断方法的一种有用的辅助检测。

AIM:To determine a species-specific real-time polymerase chain reaction ( PCR ) assay to detect Pseudomonas aeruginosa( PA) ,a secondary DNA target for PA that may provide a universal target for other bacterial pathogens,and validate both assays for diagnostic testing. METHODS:PCR detection was established against the ecfX PA gene and the 16S rRNA gene using known PA keratitis isolates. The outcome parameters for both assays were " limit of detection" ( LOD) ,amplification efficiency ( AE) ,and PAGE amplified product analysis. Both assays were validated against 20 true-positive clinical samples positive for PA DNA and 20 true-negative samples containing no PA DNA. Descriptive statistics and PAGE analysis were used as outcome parameters. RESULTS:AE of the ecfX assay was 96.6%,and LOD was 33. 6 copies of target DNA per microliter. AE of the 16S rRNA assay was 103.4%,and LOD was 8.12 copies per microliter. The sensitivity,specificity,positive predictive value,negative predictive value,and efficiency for the ecfX and 16S rRNA assays were ( 75%,95%,94%,79%,and 85%) ,and ( 70%,100%,100%,77%,and 85%) ,respectively. Both PCR assays were validated,followed by confirmation of DNA patterns from PAGE analysis. CONCLUSION:The PCR methodology described here may be a useful adjunct to standard methods in the diagnosis of PA keratitis.

R772.2

美国宾夕法尼亚州Lions Club and The Charles T. Campbell Foundation资助项目