Hilali F, Saulnier P, Chachaty E, Andremont A
Laboratoire de Microbiologie Médicale, Institut Gustave-Roussy, Villejuif, France.
Mol Biotechnol. 1997 Jun;7(3):207-16. doi: 10.1007/BF02740812.
We describe a polymerase chain reaction (PCR) that allowed detection of rRNA consensus sequences from the DNA extracted from a wide range of bacterial species in amounts as low as 10 fg. To avoid false positive results with universal primers for 16S rRNA PCR, contaminating DNA had to be eliminated from the polymerase preparations. Decontamination was undertaken before PCR to optimize treatment with DNaseI, and was followed by DNase inactivation at 94 degrees C for 50 min, which eliminated contaminating DNA at concentrations of up to 100 pg. After optimization of PCR conditions for each polymerase. Deep Vent Exo-polymerase (New England Biolabs, Beverly, MA), and super-Taq polymerase (HT Biotechnology, Cambridge, UK) were more effective than Ampli-Taq polymerase (Perkin-Elmer Cetus, Norwalk CT), Ampli-Taq LD polymerase (Perkin-Elmer Cetus) or Deep-vent polymerase (New England Biolabs). The technique described in this article might prove to be a universal method for PCR detection of small numbers of unidentified bacteria in usually sterile clinical sites, such as blood and cerebrospinal fluids, in which a broad spectrum of pathogens can be expected.
我们描述了一种聚合酶链反应（PCR），该反应能够从从多种细菌物种提取的DNA中检测出低至10 fg的rRNA共有序列。为避免16S rRNA PCR通用引物产生假阳性结果，必须从聚合酶制剂中去除污染DNA。在PCR之前进行去污处理以优化用DNaseI的处理，然后在94℃下将DNase灭活50分钟，这可消除浓度高达100 pg的污染DNA。在为每种聚合酶优化PCR条件之后。Deep Vent Exo聚合酶（新英格兰生物实验室，贝弗利，马萨诸塞州）和超级Taq聚合酶（HT生物技术公司，剑桥，英国）比Ampli-Taq聚合酶（珀金埃尔默公司，诺沃克，康涅狄格州）、Ampli-Taq LD聚合酶（珀金埃尔默公司）或Deep-vent聚合酶（新英格兰生物实验室）更有效。本文所述技术可能被证明是一种通用方法，用于在通常无菌的临床部位（如血液和脑脊液）中对少量未鉴定细菌进行PCR检测，在这些部位可能存在多种病原体。
