Silkie Sarah S, Tolcher Matthew P, Nelson Kara L
Civil and Environmental Engineering Department, University of California, Berkeley, CA 94720-1710, USA.
J Microbiol Methods. 2008 Mar;72(3):275-82. doi: 10.1016/j.mimet.2007.12.011. Epub 2007 Dec 31.
The application of real-time quantitative PCR (qPCR) for the detection of low concentrations of Escherichia coli as well as universal 16S rDNA has been hindered by false-positives due to endogenous contamination of PCR reagents with E. coli and other bacterial DNA. We optimized a DNase I decontamination method to eliminate false-positives in a qPCR assay targeting the uidA gene in E. coli. In contrast to previous methods reported in the literature, our decontamination method did not cause PCR inhibition. We determined that residual DNase I activity was the cause of the inhibition in the previous methods, and eliminated it by ensuring complete inactivation prior to qPCR. DNase inactivation was accomplished by adding dithiothreitol (DTT) and then heating for 30 min at 80 degrees C. The optimized DNase method was compared to another decontamination method, ultrafiltration, and to untreated controls. We detected contamination in 85% of the untreated commercial PCR master mix samples at a level of about 10 copies per well (12.5 microL of master mix). Both decontamination methods could eliminate up to 100 copies of added contaminant DNA and did not cause PCR inhibition, resulting in a reduction of the detection limit to 10 copies per reaction well.
实时定量PCR（qPCR）在检测低浓度大肠杆菌以及通用16S rDNA时，因PCR试剂被大肠杆菌和其他细菌DNA内源污染而出现假阳性，从而受到阻碍。我们优化了一种脱氧核糖核酸酶I（DNase I）去污方法，以消除针对大肠杆菌uidA基因的qPCR检测中的假阳性。与文献中报道的先前方法不同，我们的去污方法不会导致PCR抑制。我们确定残留的DNase I活性是先前方法中抑制现象的原因，并通过在qPCR之前确保完全失活将其消除。通过添加二硫苏糖醇（DTT）然后在80摄氏度加热30分钟来实现DNase失活。将优化后的DNase方法与另一种去污方法超滤以及未处理的对照进行比较。我们在85%的未处理商业PCR预混样本中检测到污染，污染水平约为每孔10个拷贝（12.5微升预混液）。两种去污方法都可以消除多达100个拷贝的添加污染物DNA，并且不会导致PCR抑制，从而将检测限降低至每个反应孔10个拷贝。
