Human DNA causes loss of PCR detection sensitivity and specificity

The performance and detection limits of PCR assays for the diagnosis of septic disease are strongly dependant on the method of DNA preparation. The extraction of total DNA from blood generates a mixture of human and pathogen DNA. A problem with this approach is that human DNA can provide non-specific binding sites for pathogen-specific primers. In particular, with high human to pathogen DNA ratios, non-specific primer binding can lead to false-negative and false-positive results and, hence, to a loss of sensitivity and specificity of the assay (Navarro et al. 2002). Apparent loss of specificity can be observed in a universal 16S rDNA PCR reaction as the result of the co-amplification of human sequences (Fig. 1b). In the example, a 3,230-fold mass excess of human to bacterial DNA led to complete failure in the sequence identification analysis. Furthermore, in the absence of bacterial DNA, a false-positive signal was produced by the amplification of a human gene (Fig. 1c).

Fig 1: Negative effect of human DNA on the universal 16S rDNA detection of bacterial targets.
a) 13 pg P. aeruginosa DNA (P.a.), b) mixture of P. aeruginosa DNA (13 pg) and human DNA (hum.) (42 ng), c) human DNA (42 ng).The amount of human DNA corresponds to that usually extracted from 0.2 ml blood. The amount of bacterial DNA equals approx. 2,000 cells.

Loss of sensitivity is another effect of the presence of human DNA. In the example (Fig. 2) a shift in the crossing point to approximately 3 C(T) values lower than where only the bacterial target occurred (Fig. 2, left graph). This corresponds to a detection sensitivity approximately one order of magnitude lower. The negative effect of human DNA on the sensitivity of bacterial target detection was observed over a range of more than two orders of magnitude in the amount of bacterial DNA.

Fig 2: Loss of sensitivity in the 16S rDNA detection of bacterial targets by primer binding to human DNA. Left graph: documentation of the amplification; arrows indicate the shift in C(T) values as a result of human DNA (hDNA) in the assay containing P. aeruginosa DNA (P. a.) as a target. Right graph: plot of C(T) values vs. the amount of bacterial DNA in the absence and presence of a constant amount of hDNA (42 ng).

SepsiTest™ removes human DNA

SepsiTest™ removes the human DNA during sample processing and thereby increases the sensitivity and specificity of pathogen detection. The principle is outlined in Fig. 3:  in a first step human cells are selectively lysed, followed by the degradation of the released DNA.  Bacterial and yeast cells are enriched and lysed. In the last step, the pathogen DNA is isolated.

Fig. 3: Sketch of the pre-analytic sample processing of SepsiTest™. Right image: Gelelectrophoretic demonstration of the absence of human DNA in blood extracts after SepsiTest™ treatment. For comparison a total DNA extract is shown ("QiaAmp" of Qiagen), US, unspiked blood.

SepsiTest™ excludes false PCR signals

There is a strong risk that DNA contamination can give rise to false-positive signals in highly sensitive PCR assays targeting conserved bacterial sequences. This can occur during the procedure, but it can also come from the contamination of the working materials with DNA from numerous exogenous sources. Furthermore, a prominent source of background DNA is the PCR mastermix itself. In the first place, the polymerising enzyme can contain traces of DNA from the production strain. But primers and dNTPs can also be contaminated with bacterial DNA. The problem of DNA contamination becomes particularly problematical when bacteria are present in small numbers.

Molzym has developed a technology that efficiently removes traces of DNA from PCR mastermix components and, thus, guarantees exclusion of false-positive signals. With SepsiTest™, a product is offered that allows reliable and sensitive detection of pathogens with the highest sensitivity issues in clinical samples. Strong signals in the assay containing target DNA allows the detection of small numbers of pathogens, for instance 40 S. aureus cfu/ml  blood.

SepsiTest™ can be run on essentially all PCR-machines including Roche LightCycler, ABI StepOne, Stratagene Mx3000P and Biorad Opticon

References:

E. Navarro, J. Escribano, J.A. Fernández, J. Solera (2002). Comparison of three different PCR methods for detection of Brucella spp. in human blood samples. FEMS Immunol. Med. Microbiol. 34: 147-151.