Obstacles of Multiplex Real-Time PCR for Bacterial 16S rDNA: Primer Specifity and DNA Decontamination of Taq Polymerase.

ABSTRACT: BACKGROUND: The detection of a broad range of bacteria by PCR is applied for the screening of blood and blood products with special attention to platelet concentrates. For practical use it is desirable that detection systems include Gram-positive, Gram-negative and non-Gram-stainable bacteria. It is quite challenging to achieve high sensitivity along with a clear negative control with PCR reagents, because especially Taq polymerase is contaminated with traces of bacterial DNA. METHODS: Bacterial DNA decontamination of Taq polymerase was attempted by two different methods using the restriction enzyme Sau 3A1 and microfiltration. Additionally a commercially available Taq polymerase depleted of bacterial DNA was included. A published real-time PCR specific for Gram-negative bacteria was adapt

SUBMITTER: Philipp S 

PROVIDER: S-EPMC2914405 | biostudies-literature | 2010 Feb

REPOSITORIES: biostudies-literature