Direct DNA amplification from crude clinical samples using a PCR enhancer cocktail and novel mutants of Taq

Abstract

PCR-based clinical and forensic tests often have low sensitivity or even false-negative results caused by potent PCR inhibitors found in blood and soil. It is widely accepted that purification of target DNA before PCR is necessary for successful amplification. In an attempt to overcome PCR inhibition, enhance PCR amplification, and simplify the PCR protocol, we demonstrate improved PCR-enhancing cocktails containing nonionic detergent, l-carnitine, d-(+)-trehalose, and heparin. These cocktails, in combination with two inhibitor-resistant Taq mutants, OmniTaq and Omni Klentaq, enabled efficient amplification of exogenous, endogenous, and high-GC content DNA targets directly from crude samples containing human plasma, serum, and whole blood without DNA purification. In the presence of these enhancer cocktails, the mutant enzymes were able to tolerate at least 25% plasma, serum, or whole blood and as high as 80% GC content templates in PCR reactions. These enhancer cocktails also improved the performance of the novel Taq mutants in real-time PCR amplification using crude samples, both in SYBR Green fluorescence detection and TaqMan assays. The novel enhancer mixes also facilitated DNA amplification from crude samples with various commercial Taq DNA polymerases.

Figure 1

Effect of PEC on PCR specificity and sensitivity. A: The impact of PEC on PCR specificity was assessed by directly amplification of the HRES1 target from blood samples in the absence (W/O) or presence (W/W) of the enhancer; 1.3 mol/L of betaine was included for comparison. The blood sample was serially fivefold diluted from 4 to 0.00001% (final concentration in PCR, lanes 1 to 9), and the negative control contained no DNA or blood (N). A 488-bp HRES1 target (GC content is 81%) was amplified from different concentrations of blood sample. OT, OmniTaq; OKT, Omni Klentaq. B: PCR sensitivity in the blood sample was also evaluated by amplification of the same target from both purified DNA and blood. The blood sample was serially fivefold diluted from 4 to 0.00001% (final concentration in PCR, lanes 1 to 9), and a 488-bp HRES1 target was amplified both from purified DNA and blood containing the same amount of DNA in presence of PEC. The negative control contained no DNA or blood (N). PCR products were resolved in a 1.5% agarose gel. Lanes M, DNA standards ladder.

Figure 2

Tolerance of OmniTaq (OT) and Omni Klentaq (OKT) to blood treated with different anticoagulants. Two endogenous targets, 300 bp of the β-actin gene (A) and 488 bp of the HRES1 gene (B), were amplified from 1.25 to 25% (of total PCR volume) from a whole blood sample treated with citrate, heparin, or EDTA, separately, using OT or OKT in the absence (W/O) or presence (W/W) of PEC. The positive control contained 4 ng of purified DNA (0). The products were analyzed in 1.5% agarose gel electrophoresis. Lanes M, DNA standards ladder.

Figure 3

Performance comparison of OT and OKT versus commercial enzymes and their PCR enhancers in amplification of GC-rich targets. The gene targets of SIM2 (1202 bp, GC content 75%, lane 1), DIP2A (1185 bp, GC content 71%, lane 2), and SLC19A (980 bp, GC content 74%, lane 3) were amplified from purified DNA or 5% whole blood (final concentration in PCR) in the absence (A) or in the presence (B) of PCR enhancers. FastStart Taq was combined with GC Solution, HotStarTaq Plus with Q-Solution, plain Taq with Hi Spec Additive, and OT and OKT with PEC. A negative control contained no DNA and blood (N). The position of the specific products 1 to 3 is shown by arrows.

Figure 4

Application of PEC-Plus in PCR in plasma and serum. A 189-bp target of mousepox virus (EV107 gene) was amplified by the OT/OKT mix (1:1) from 0.1 ng of viral DNA and crude samples containing virus particles, including 10% whole blood, serum, or plasma (final concentration in PCR) treated with heparin, citrate, and EDTA. The reactions were performed in the absence of enhancer or in the presence of heparin alone, a combination of heparin and trehalose, PEC, and PEC-Plus (PEC supplemented with heparin). Negative controls contained no virus DNA (N). Lane 1, DNA; lane 2, heparin-treated plasma; lane 3, citrate-treated plasma; lane 4, EDTA-treated plasma; lane 5, serum; and lane 6, heparin-treated blood. Lanes M, DNA standards ladder.

Figure 5

Application of PEC in SYBR Green real-time PCR. A master mixture containing 5% whole blood (in total PCR volume) was made, and aliquots of it were distributed to PCR tubes. The supernatant of the HVS2 cell culture was fivefold serially diluted with water, from which dilutions of 1 μl were added to each reaction. A 137-bp HVS2 target gene was amplified from these samples using OT (A and B) or OKT (C and D) and SYBR green detection in the presence (A and C) or absence of PEC (B and D). PCR was performed for 40 cycles in an Option 2 real-time cycler in the presence of 10× SYBR Green. A negative control contained no virus. The Ct values for the panels where the enzymes were functional (A, C, and D) are provided at the bottom.

Figure 6

Application of PEC in a real-time PCR TaqMan assay. A qPCR Master Mix containing 10% human plasma (10% of total PCR volume) was made and aliquoted to the PCR tubes. A mousepox virus cell culture supernatant was 10-fold serially diluted with water and a 189-bp mousepox virus gene (EV107 gene) was amplified from these crude samples. A parallel sample series contained equivalent amounts of purified virus DNA dilutions. An OT/OKT enzyme mixture (1:1) was used and the reactions contained PEC (A and B). The same virus target was also amplified with AmpliTaq Gold as a comparison (C and D). PCR was performed for 45 cycles in an Option 2 real-time cycler, and the amplification was detected with 200 nmol/L TaqMan probe. A negative control contained no virus or DNA. The Ct values are shown at the bottom.

Figure 7

qPCR assessment of sensitivity of pathogen detection in crude samples containing whole blood. Ten-fold serial dilutions of mousepox virus were made in 100 μl of heparinized blood (final virus concentration ranged from 7.2 × 10⁵ to 0.72 copies/μl). These virus-spiked blood samples were split in half and virus DNA was purified from 50 μl of each sample with a QIAGEN DNA extraction kit (A). DNA purified from these samples was finally eluted in 50 μl of water, thus restoring the original blood volume. The other half of the crude blood samples was directly used in PCR (B), and 1.25 μl of each purified DNA and blood sample series (5% final volume in PCR) were used in parallel to amplify a 189-bp mousepox virus target (EV107 gene), using Omni Taq and SYBR Green detection in the presence of PEC. A negative control contained no virus or DNA. The concentrations of the virus particles and the corresponding Ct values are shown at the bottom. With both purified DNA and blood seven virus copies/μl were detected (A and B and curves 6).