MPD enhanced quantitative PCR (qPCR/MPD): One of the greatest challenges of biomedical diagnostics is the improvement of assay systems for agents such as human immunodeficiency virus (HIV), hepatitis virus and other viruses. Also, supersensitive DNA/RNA detection and quantitation methods have been increasingly used in such fields as food quality, forensics and "counter terrorism". The MPD technique enables substantial improvements to the sensitivity, accuracy, and reproducibility of DNA diagnostic tests. MPD instrumentation permits the reduction of non-specific biological background (NSBB). We have developed a high sensitivity/high throughput method using microtiter format.
The large number of amplification cycles (30-50) required, the variability in efficiency of amplification, variability in the behavior of standards from reaction to reaction, the accumulation of NSBB and heteroduplex formation all make quantitative PCR very difficult. The limit of quantitation is dictated by the sensitivity of detection systems. The current limit of reliable quantitation is about 50 copies of DNA. The applications of qPCR, including real time PCR from Perkin Elmer, are limited by relatively high cost of appropriate instrument (ca. $100,000), relatively long assay time ( 3 hours per test) and low reproducibility for less than 50 copies of DNA.
Current PCR at very low number of copies requires a large number, say up to 45, cycles of PCR. However, for more than 25 cycles the amplification efficiency considerably decreases. This leads to serious artifacts and the reliability of quantitation is diminished. Methods based on the use of mimics and Taqman have been developed, however, even in this case the quantitation is not reliable for less than 50 copies. Also, the need to use a very large number of amplification cycles makes the selection of primers more difficult, because of increased mispriming. The specificity of PCR is diminished due to the above said mispriming.
We demonstrated that more sensitive detection enabled by MPD considerably facilitates quantitative PCR. We typically use about 20 cycles of PCR even for less than 10 copies of DNA. We demonstrated reliable detection of a single copy HIV-1 provirial DNA. We demonstrated qPCR/MPD of the HIV-1 gag gene from 6 to a few thousand copies per milliliter of serum. We achieved reliable qPCR in the presence of up to 8 micrograms of human DNA and in PBMC samples. Similar results were achieved for CMV and RSV.
We developed reliable methods for quantitation of a few bacteria. As examples we selected bacteria responsible for food borne diseases, i.e. Salmonella and pathogenic E. coli. Both the limit of quantitation (less than 10 copies) and reproducibility are better than using prior art methods. We also simplified the sample handling considerably by developing new methods permittting DNA quantitation without DNA extraction.
We developed qPCR/MPD that can measure less than 10 copies (the lower limit is set by Poisson statistics) to a few thousands of copies from a single dilution of sample. This assay uses 125I-dCTP labeling and has been adapted to streptavidin capture of biotinylated PCR products on 96 well microtiter plates. We implemented a capture technique which uses two distinct labels for mimic and for target, respectively. It allows measurement of target and mimic simultaneously in the same well. This methodology is both more reliable and faster than current methods. Applications of this new technique for microbial detection and quantitation is one of the important strategic objectives of BioTraces. In food quality testing use of our DNA analysis method enables fast and reliable diagnosis of many important food pathogens.
We addressed the need for an integrated diagnostics system that provides the most sensitive, low cost and rapid tests. MPD technology for DNA quantitation permits the accomplishment of a few copies sensitivity; high rapidity (assay time is below 1 hour), and high throughput via automation. This includes miniaturization and semi-automation permitting parallel testing of 96 samples. A system for concurrent quantitation of 384 samples has been developed.
Results: We have developed a reliable, MPD enhanced qPCR method for quantitating rare DNAs (qPCR/MPD), i.e. for less than 10 copies of a given target. In the following we illustrate the ultra-sensitive qPCR/MPD assay for virial targets such as HIV-1 and CMV (see Figure 1 and Figure 2)
The MPD enhanced qPCR can be optimized for different ranges of interest by manipulating the number of qPCR cycles and the concentration of an internal calibrator. The mimic concentration should fall within a factor of 100 of the number of copies in the unknown sample. Our results suggest that use of about 20 PCR cycles improves the dynamic range of quantitation. We achieved quantitation of a few copies of HIV-1, CMV, RSV, Salmonella, E. coli, and kanamycin resistance gene in the presence of up to 10 micrograms of background human genomic DNA. Using MPD readout, standard curves were generated reliably over the range of 6-6,000 copies of target DNA sequences using a single concentration of mimic. Thus, MPD enhanced quantitative PCR with internal controls permits reliable quantitation of DNA at a level ten fold lower than prior-art methods.
It is important to stress that the excellent sensitivity, linearity and specificity of qPCR/MPD are all due to the same fact: the unsurpassed sensitivity of MPD. Even for a few copies of DNA, a relatively small number of cycles of PCR are used. The main advantage of using MPD is that qPCR/MPD never approaches saturation of the PCR process.
One of the main difficulties of using PCR in food quality testing is the presence of inhibitors, proteases and nucleases which interfere destructively with amplification. This may lead to artifacts and requires the development of specialized DNA extraction methods. We studied this problem for the case of milk and diary products, and developed a procedure permitting the reliable quantitation of nucleic acids (in our case HIV-1 gag provirial DNA in milk) without DNA extraction. We are currently performing a similar studies for other targets.
MPD enhanced quantitative PCR for HIV-1 (qPCR/MPD): First, we developed a gel based competitive PCR based on radioactive nucleotide incorporation. Second, we replaced the gel analysis with a DNA capture step and replaced the labeled nucleotide with labeled primers. Thus, we implemented user-friendly qPCR/MPD in microtiter format. We are now working on lowering the detection limit, i.e., moving towards implementation of MPD enhanced direct detection of DNA(dqDNA/MPD).
The number of unknown copies is read from the standard curve or calculated using a regression fit curve defined with 95% confidence limits. For 6.25 copies nominal, qPCR/MPD yields 5.64 copies and a 95% confidence interval of 4.18 - 8.55 copies. This is an excellent result given the statistical uncertainty in this experiment. The results confirm that the analytical limit is approaching a single copy. In other experiments, a copy number of 27.8 was calculated for the nominal input mimic concentration of 25 copies. A 95% confidence interval gives values ranging between 20 to 30 copies. This means that the measurement of the copy number in an unknown sample against this standard curve in one hundred qPCRs run in parallel, would result in only five results outside of the 20 - 30 copy number interval. Better than 20% reproducibility is achieved for 40 samples, each containing nominally 25 copies of the target DNA. For 100 copies of target DNA/samples, the reproducibility is about 15%.
The linearity of qPCR/MPD is presented in Figure 3. Note the excellent linearity down to ten copies of HIV-1 target. We also note, that the results using qPCR/MPD in gel and microtiter formats are practically identical (see Figure 4). We note the excellent reproducibility of qPCR/MPd (see Figure 5 and Figure 6) for nominal number of copies of 32 and 112, respectively).<
Simplified sample handling: We demonstrated high reliabilty of qPCR/MPD even in presence of large amount, up to 8 microgram, of other DNA, see Figure 7. Note that difference between runs is higher than dependence on amount of DNA.
PCR based DNA diagnostics from clinical samples is difficult to perform due to biological matrix artifacts, i.e. due to resident proteases and other non-specific PCR inhibitors. This introduces the need for costly pre PCR sample handling procedures,e.g. extraction of DNA before amplification. But extraction of trace amounts of DNA can lead to variability of recovery and thus loss of quantitative accuracy. Since most inhibitors are deleterious only at high concentrations, we sought to minimize the effects of inhibitors by simple dilution. The appropriate experiments were performed for HIV-1 gag in milk and blood.
In brief, milk (2% fat) was spiked with 500 copies per microliter of HIV-1 gag proviral DNA. Varying dilutions were added to 50 microliter PCR reactions. If 20 microliters of milk was used, no target or mimic bands were observed, but the same amount of milk diluted ten-fold gave perfectly good amplifications. Thus, the inhibitory effect of milk on PCR is overcome by ten-fold dilution. Because the qPCR/MPD is fully quantitative down to a few copies of DNA, the use of ten-fold dilution permits quantitation without DNA extraction down to about 50 copies/ml of unprocessed material, e.g. milk.
Similar experiments were performed in other physiological fluids, including a series on HIV-1 gag measurements in serum. In all these experiments, qPCR/MPD permitted much better reliability than current methods. The qPCR/MPD has been demonstrated to be insensitive to diverse interfering processes, e.g. presence of polymerase inhibitors. This advantage is due to the small number of amplification steps used.
RNA targets: We extented our study to MPD enhanced RT-PCR (qRT-PCR/MPD). The excellent linearity and reproducibility of qRT-PCR/MPD for RNA encoding PSMTA is illustrated in Figure 8.
MPD enabled high throughput multiplex quantitative PCR (mqPCR/MPD): We developed a qPCR/MPD in microtiter format for both HIV-1 gag and bacterial targets (Salmonella and pathogenic E. coli). Herein, a brief review of the results for Salmonella is provided. We compared results obtained by the gel method to the microtiter capture method (see Figure 9). A single set of cold PCR reactions corresponding to a dilution series of mimic was labeled first by using the same primers for labeling as were used for the cold PCR and second by using a nested biotinylated primer with one of the original primers (semi-nested method). The first set was quantified with the MPD scanner after gel electrophoresis and the second set was quantified by counting the individual microwells in the ssMPD. A further enhancement to the labeling PCR step was obtained by using fully nested primers with a mimic primer labeled by 32P and a target primer labeled with 125I. The principle of this method was established through the labeling of an amino-derivatized oligonucleotide with radioactively labeled Bolton-Hunter reagent. The dual label qPCR/MPD was performed. We demonstrated the excellent linearity and reproducibility of the data.
Similar data had been obtained for pathogenic
E.coli 0157:H7. Figure 10 shows the excelent
linearity
both in microtiter format.
Figure 11 shows the high reproducibility; our CV is
about 20%
at nominal 64 copies.
The high sensitivity of MPD permits the multiplex quantitative PCR (mqPCR/MPD).
The data see Figure 12 and Figure 13,
demonstrate that
linearity of mqPCR/MPD is down to a few copies. Furthermore, even when the
second target has orders of magnitude different abundance, there is no visible
influence of quantitation.
SUMMARY: We have developed a highly sucessful MPD-enhanced quantitative PCR assay. We developed both gel based and microtiter formatted assays which permit reliable quantitation at a few copies of DNA. Not only the sensitivity, but also the ease of sample preparation and improved rejection of biological matrix artifacts, makes qPCR/MPD a superior method for biomedical diagnostics and food quality testing.