Validation of PCR for the detection of Pseudomonas aeruginosa from corneal samples

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.

使用PCR检测角膜绿脓杆菌(英文)

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。

铜绿假单胞菌实时荧光重组酶介导链替换核酸扩增方法建立及应用

目的建立一种快速检测铜绿假单胞菌的实时荧光重组酶介导链替换核酸扩增(real-time RAA)方法。方法基于铜绿假单胞菌ecfX基因设计特异性引物和exo探针,通过灵敏性、特异性和疑似菌株检测评估所建立方法的有效性。结果Real-time RAA方法在39℃等温条件下反应20 min即可实现对铜绿假单胞菌的有效检测;特异性强,仅对铜绿假单胞菌出现特异性扩增;灵敏性高,对铜绿假单胞菌基因组DNA的检出限为3.0×10^3 fg/反应,对纯培养铜绿假单胞菌的检出限为1.0×10^3 CFU/反应。应用所建立的real-time RAA方法对分离的36株疑似铜绿假单胞菌进行检测,结果均为铜绿假单胞菌,同VITEK 2 Compact生化鉴定方法和real-time PCR方法结果一致。结论本试验所建立的real-time RAA方法反应快速、操作简单、可靠性高,可用于不同来源的铜绿假单胞菌的快速检测和鉴定。