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 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.

Fig. 1

Positioning of a the forward- and b reverse primers: The sequence of parts of the 16S rRNA gene of E. coli is shown. Genetic differences to Mycobacteriae (gray) and Staphylococci (bold) are shown. Homologous regions of human mitochondrial DNA are displayed for illustration. Modifications of primers T lead to an improved detection of mycobacterial DNA (primer 16S-Mtb-F and -R, gray) and gram-positive bacteria (primer 16S-Staph-F and -R, bold).

Fig. 2

Exemplary amplification plot of different E. coli dilutions. Amplification plots of 1,000, 500, 100, 50 CFU per PCR and negative control (no template control) for PCR cycles 25 to 45 are shown. C(t) = 27.90, C(t) = 29.08, C(t) = 31.21 and C(t) = 33.30 represent 1,000, 500, 100, and 50 CFU/PCR, respectively. C(t) = 34.21 represents the negative control.