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

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 adapted for Gram-positive bacteria, including certain Staphylococcus species and Mycobacteria, and was used to charge the three Taq polymer-ases depleted of bacterial DNA contamination Results: Despite published reports about successful DNA decontamination, all three approaches performed poorly in experiments done in this study. Sensitivity ranged at approximately 50–100 colony forming units (CFU) per PCR reaction for Escherichia coli and Staphylococcus epidermidis, corresponding to 1,250–2,500 CFU/ml sample material. Conclusion: It seems unsatisfying to accept detection limits that high for diagnostic bacterial PCR even if highly multiplexed. Reliable methods for DNA decontamination of Taq polymerase are needed and would present one important step towards bacterial DNA detection with high sensitivity.

Expanding Possibilities of Rolling Circle Amplification as a Biosensing Platform

“…[36][37][38] Therefore, great attention is paid in an alternative rapid and convenient method for the detection of living microbes that can overcome the technical and economic difficulties of conventional cultivation-and PCR-based methods for microbial detection.…”

Development and evaluation of a TaqMan duplex real-time PCR quantification method for reliable enumeration of Candidatus Microthrix

“…Secondly, the main problem for detection of bacterial DNA is the achievement of a high sensitivity along with a clear negative control. Up to now, this is an unresolved problem since the reagents used in the PCR process are contaminated with trace amounts of bacterial DNA, especially the Taq polymerase, where contamination originates from its production in bacterial cultures (Philipp et al, 2010). Therefore, the use of functional genes for multiplex PCR seems to be much easier.…”

A Novel, Universal and Sensitive Lateral-Flow Based Method for the Detection of Multiple Bacterial Contamination in Platelet Concentrations

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