A Mechanistic Model of PCR for Accurate Quantification of Quantitative PCR Data

Background

Quantitative PCR (qPCR) is a workhorse laboratory technique for measuring the concentration of a target DNA sequence with high accuracy over a wide dynamic range. The gold standard method for estimating DNA concentrations via qPCR is quantification cycle () standard curve quantification, which requires the time- and labor-intensive construction of a standard curve. In theory, the shape of a qPCR data curve can be used to directly quantify DNA concentration by fitting a model to data; however, current empirical model-based quantification methods are not as reliable as standard curve quantification.

Principal Findings

We have developed a two-parameter mass action kinetic model of PCR (MAK2) that can be fitted to qPCR data in order to quantify target concentration from a single qPCR assay. To compare the accuracy of MAK2-fitting to other qPCR quantification methods, we have applied quantification methods to qPCR dilution series data generated in three independent laboratories using different target sequences. Quantification accuracy was assessed by analyzing the reliability of concentration predictions for targets at known concentrations. Our results indicate that quantification by MAK2-fitting is as reliable as standard curve quantification for a variety of DNA targets and a wide range of concentrations.

Significance

We anticipate that MAK2 quantification will have a profound effect on the way qPCR experiments are designed and analyzed. In particular, MAK2 enables accurate quantification of portable qPCR assays with limited sample throughput, where construction of a standard curve is impractical.     

LORD-Q: a long-run real-time PCR-based DNA-damage quantification method for nuclear and mitochondrial genome analysis

DNA damage is tightly associated with various biological and pathological processes, such as aging and tumorigenesis. Although detection of DNA damage is attracting increasing attention, only a limited number of methods are available to quantify DNA lesions, and these techniques are tedious or only detect global DNA damage. In this study, we present a high-sensitivity long-run real-time PCR technique for DNA-damage quantification (LORD-Q) in both the mitochondrial and nuclear genome. While most conventional methods are of low-sensitivity or restricted to abundant mitochondrial DNA samples, we established a protocol that enables the accurate sequence-specific quantification of DNA damage in >3-kb probes for any mitochondrial or nuclear DNA sequence. In order to validate the sensitivity of this method, we compared LORD-Q with a previously published qPCR-based method and the standard single-cell gel electrophoresis assay, demonstrating a superior performance of LORD-Q. Exemplarily, we monitored induction of DNA damage and repair processes in human induced pluripotent stem cells and isogenic fibroblasts. Our results suggest that LORD-Q provides a sequence-specific and precise method to quantify DNA damage, thereby allowing the high-throughput assessment of DNA repair, genotoxicity screening and various other processes for a wide range of life science applications.     

Quantification of PCR products by phosphate measurement

Various techniques for quantification of PCR are available. Most frequently, the densitometric intensities of ethidium bromide-stained PCR products separated in gels are compared after normalizing to the levels of housekeeping gene products such as β-actin. More precise, but extremely time consuming, is the technique of competitive PCR. Newer methods, such as tracking amplification in real-time, have high start-up and maintenance costs (e.g., TaqMan, Applied Biosystems; LightCycler, Roche; I-Cycler, Bio-Rad). Here, I describe an alternative, simple technique to quantify PCR products by determining the entire phosphate released during PCR. The method can be performed using common laboratory equipment, and the reagents needed are extremely cheap. The method is validated by measuring the induction of inducible nitric oxide synthase gene expression in cell culture and comparing the results with data obtained by LightCycler experiments and RNase protection assays.     

Quantification of Leishmania infantum parasites in tissue biopsies by real-time polymerase chain reaction and polymerase chain reaction-enzyme-linked immunosorbent assay

Most of the experimental studies of Leishmania spp. infection require the determination of the parasite load in different tissues. Quantification of parasites by microscopy is not very sensitive and is time consuming, whereas culture microtitrations remain laborious and can be jeopardized by microbial contamination. The aim of this study was to quantify Leishmania infantum parasites by real-time polymerase chain reaction (PCR) using specific DNA TaqMan probes and to compare the efficacy of detection of this technique with a PCR-enzyme-linked immunosorbent assay (ELISA). For this purpose, spleen and liver samples from L. infantum-infected mice were collected during a 3-mo longitudinal study and analyzed by both methods. PCR-ELISA failed to quantify Leishmania spp. DNA in samples with very low or very high numbers of parasites. Real-time PCR was more sensitive than PCR-ELISA, detecting down to a single parasite, and enabled the parasite quantification over a wide, 5-log range. In summary, this study developed a method for absolute quantification of L. infantum parasites in infected organs using real-time TaqMan PCR.     

Rapid competitive PCR using melting curve analysis for DNA quantification.

A rapid competitive PCR method was developed to quantify DNA on the LightCycler. It rests on the quantitative information contained in the melting curves obtained after amplification in the presence of SYBR Green I. Specific hybridization probes are not required. Heterologous internal standards sharing the same primer binding sites and having different melting temperatures to the natural PCR products were used as competitors. After a co-amplification of known amounts of the competitor with a DNA-containing sample, the target DNA can be quantified from the ratio of the melting peak areas of competitor and target products. The method was developed using 16S rDNA fragments from Streptococcus mutans and E. coli and tested against existing PCR-based DNA quantification procedures. While kinetic analysis of real-time PCR is well established for the quantification of pure nucleic acids, competitive PCR on the LightCycler based on an internal standardization was found to represent a rapid and sensitive alternative DNA quantification method for analysis of complex biological samples that may contain PCR inhibitors.     

Detection of telomerase activity utilizing energy transfer primers: comparison with gel- and ELISA-based detection

We have developed a closed-tube format telomeric repeat amplification protocol (TRAP) assay for direct quantification of telomerase activity within the PCR vessel. The assay utilizes energy transfer (ET) primers, which emit fluorescence only upon incorporation into PCR products. This novel ET primer system (Amplifluor primers) has major advantages over existing detection methods because it eliminates the need for post-PCR processing and thus reduces greatly the risk of carryover contamination and the time required for the sample analysis. The assay is as sensitive, specific and quantitative as the polyacrylamide gel-based or ELISA-based TRAP assay.     

Real-time PCR detection and quantification of fish probiotic Phaeobacter strain 27-4 and fish pathogenic Vibrio in microalgae, rotifer, Artemia and first feeding turbot (Psetta maxima) larvae

Aims:  To develop a SYBR Green quantitative real-time PCR protocol enabling detection and quantification of a fish probiotic and two turbot pathogenic Vibrio spp. in microcosms.
Methods and Results:  Phaeobacter 27-4, Vibrio anguillarum 90-11-287 and Vibrio splendidus DMC-1 were quantified as pure and mixed cultures and in presence of microalgae ( Isochrysis galbana ), rotifers ( Brachionus plicatilis ), Artemia nauplii or turbot ( Psetta maxima ) larvae by real-time PCR based on primers directed at genetic loci coding for antagonistic and virulence-related functions respectively. The optimized protocol was used to study bioencapsulation and maintenance of the probiont and pathogens in rotifers and for the detection and quantification of Phaeobacter and V. anguillarum in turbot larvae fed rotifers loaded with the different bacteria in a challenge trial.
Conclusions:  Our real-time PCR protocol is reproducible and specific. The method requires separate standard curve for each host organism and can be used to detect and quantify probiotic Phaeobacter and pathogenic Vibrio bioencapsulated in rotifers and in turbot larvae.
Significance and Impact of the Study:  Our method allows monitoring and quantification of a turbot larvae probiotic bacteria and turbot pathogenic vibrios in in vivo trials and will be useful tools for detecting the bacteria in industrial rearing units.     

Species-specific real-time PCR cell number quantification of the bloom-forming cyanobacterium Planktothrix agardhii

A species-specific method to detect and quantify Planktothrix agardhii was developed by combining the SYBR Green I real-time polymerase chain reaction technique with a simplified DNA extraction procedure for standard curve preparation. Newly designed PCR primers were used to amplify a specific fragment within the rpoC1 gene. Since this gene exists in single copy in the genome, it allows the direct achievement of cell concentrations. The cell concentration determined by real-time PCR showed a linear correlation with the cell concentration determined from direct microscopic counts. The detection limit for cell quantification of the method was 8?cells?μL(-1), corresponding to 32 cells per reaction. Furthermore, the real-time qPCR method described in this study allowed a successful quantification of P. agardhii from environmental water samples, showing that this protocol is an accurate and economic tool for a rapid absolute quantification of the potentially toxic cyanobacterium P. agardhii.     

The SSV-Seq 2.0 PCR-Free Method Improves the Sequencing of Adeno-Associated Viral Vector Genomes Containing GC-Rich Regions and Homopolymers

Adeno-associated viral vectors (AAV) are efficient engineered tools for delivering genetic material into host cells. The commercialization of AAV-based drugs must be accompanied by the development of appropriate quality control (QC) assays. Given the potential risk of co-transfer of oncogenic or immunogenic sequences with therapeutic vectors, accurate methods to assess the level of residual DNA in AAV vector stocks are particularly important. An assay based on high-throughput sequencing (HTS) to identify and quantify DNA species in recombinant AAV batches is developed. Here, it is shown that PCR amplification of regions that have a local GC content >90% and include successive mononucleotide stretches, such as the CAG promoter, can introduce bias during DNA library preparation, leading to drops in sequencing coverage. To circumvent this problem, SSV-Seq 2.0, a PCR-free protocol for sequencing AAV vector genomes containing such sequences, is developed. The PCR-free protocol improves the evenness of the rAAV genome coverage and consequently leads to a more accurate relative quantification of residual DNA. HTS-based assays provide a more comprehensive assessment of DNA impurities and AAV vector genome integrity than conventional QC tests based on real-time PCR and are useful methods to improve the safety and efficacy of these viral vectors.     

Quantification of Mature MicroRNAs Using Pincer Probes and Real-Time PCR Amplification

The robust and reliable detection of small microRNAs (miRNAs) is important to understand the functional significance of miRNAs. Several methods can be used to quantify miRNAs. Selectively quantifying mature miRNAs among miRNA precursors, pri-miRNAs, and other miRNA-like sequences is challenging because of the short length of miRNAs. In this study, we developed a two-step miRNA quantification system based on pincer probe capture and real-time PCR amplification. The performance of the method was tested using synthetic mature miRNAs and clinical RNA samples. Results showed that the method demonstrated dynamic range of seven orders of magnitude and sensitivity of detection of hundreds of copies of miRNA molecules. The use of pincer probes allowed excellent discrimination of mature miRNAs from their precursors with five Cq (quantification cycle) values difference. The developed method also showed good discrimination of highly homologous family members with cross reaction less than 5%. The pincer probe-based approach is a potential alternative to currently used methods for mature miRNA quantification.     

Development of matrix lysis for concentration of gram positive bacteria from food and blood

The development of a fast, reliable and inexpensive protocol for the concentration of bacteria from food by the removal of fat, carbohydrates and proteins that is compatible with downstream alternative DNA-based quantification methods is described. The protocol was used for dairy products, cooked and smoked fish and meat, carbohydrate-rich cooked products, ready-to-eat sauces, egg and blood. Lysis resulted in pellets of reasonable size for further processing. Starch, plant materials, fungi, tissues such as sinew, and chalaza could not be dissolved. Using L. monocytogenes, S. aureus and B. cereus as model organisms, microscopic analysis of the remaining bacterial pellets revealed that the recovered bacteria remained physically intact, albeit that the viability of the cells was compromised. Using real-time PCR, 7.3 CFU of L. monocytogenes were detected in artificially contaminated ultra-high temperature treated (UHT) milk and raw milk.     

Detection of 2-hydroxyethidium in cellular systems: a unique marker product of superoxide and hydroethidine

Various detection methods of the specific product of reaction of superoxide (O(2)(*-)) with hydroethidine (HE), namely 2-hydroxyethidium (2-OH-E(+)), and with its mitochondria-targeted analog are described. The detailed protocol for quantification of 2-OH-E(+), the unique product of HE/O(2)(*-) in cellular systems, is presented. The procedure includes cell lysis, protein precipitation using acidified methanol and HPLC analysis of the lysate. Using this protocol, we determined the intracellular levels of 2-OH-E(+) and E(+) in the range of 10 and 100 pmol per mg protein in unstimulated macrophage-like RAW 264.7 cells. In addition to HE, 2-OH-E(+) and E(+), we detected several dimeric products of HE oxidation in cell lysates. As several oxidation products of HE are formed, the superoxide-specific product, 2-OH-E(+) needs to be separated from other HE-derived products for unequivocal quantification.     

Enumeration of total bacteria and bacteria with genes for proteolytic activity in pure cultures and in environmental samples by quantitative PCR mediated amplification

Real-time quantitative PCR assays were developed for the absolute quantification of different groups of bacteria in pure cultures and in environmental samples. 16S rRNA genes were used as markers for eubacteria, and genes for extracellular peptidases were used as markers for potentially proteolytic bacteria. For the designed 16S rDNA TaqMan assay, specificity of the designed primer-probe combination for eubacteria, a high amplification efficiency over a wide range of starting copy numbers and a high reproducibility is demonstrated. Cell concentrations of Bacillus cereus, B. subtilis and Pseudomonas fluorescens in liquid culture were monitored by TaqMan-PCR using the 16S rDNA target sequence of Escherichia coli as external standard for quantification. Results agree with plate counts and microscopic counts of DAPI stained cells. The significance of 16S rRNA operon multiplicity to the quantification of bacteria is discussed.Furthermore, three sets of primer pair together with probe previously designed for targeting different classes of bacterial extracellular peptidases were tested for their suitability for TaqMan-PCR based quantification of proteolytic bacteria. Since high degeneracy of the probes did not allow accurate quantification, SybrGreen was used instead of molecular probes to visualize and quantify PCR products during PCR. The correlation between fluorescence and starting copy number was of the same high quality as for the 16S rDNA TaqMan assay for all the three peptidase gene classes. The detected amount of genes for neutral metallopeptidase of B. cereus, for subtilisin of B. subtilis and for alkaline metallopeptidase of P. fluorescens corresponded exactly to the numbers of bacteria investigated by the 16S rDNA targeting assay.The developed assays were applied for the quantification of bacteria in soil samples.     

Detection and Quantification of Dehalococcoides-Related Bacteria in a Chlorinated Ethene-Contaminated Aquifer Undergoing Natural Attenuation

Detection and quantification of bacteria related to Dehalococcoides is essential for the development of effective remediation strategies for tetrachloroethene (PCE)-contaminated sites. In this study, the authors applied three methods for quantifying Dehalococcoides-like bacteria in a PCE-contaminated aquifer undergoing natural attenuation in Grenchen, Switzerland: a catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH) protocol, a competitive nested polymerase chain reaction (PCR) approach, and a direct PCR end point quantification with external standards. For the investigated aquifer, multiple lines of evidence indicated that reductive dechlorination (and likely dehalorespiration) was an active process. Both PCR-based quantification methods indicated that low numbers of mostly sediment-bound Dehalococcoides were present in the contaminated zone of the Grenchen aquifer. Estimates based on the quantitative PCR methods ranged from 2.1 × 10⁷ to 1.5 × 10⁸ sediment-bound Dehalococcoides 16S rRNA gene copies per liter of aquifer volume. In contrast, the liquid phase only contained between 8 and 80 copies per liter aquifer volume. CARD-FISH was not sensitive enough for the quantification of Dehalococcoides cell numbers in this aquifer. Cloning and sequencing of the PCR products revealed the presence of sequences closely related to Dehalococcoides isolates such as D. ethenogenes and Dehalococcoides sp. BAV1. An apparently abundant group (termed “Grenchen Cluster”) of sequences more distantly related to Dehalococcoides was also identified, so far without cultured representatives.     

Comparison of relative mRNA quantification models and the impact of RNA integrity in quantitative real-time RT-PCR

Relative quantification in quantitative real-time RT-PCR is increasingly used to quantify gene expression changes. In general, two different relative mRNA quantification models exist: the delta-delta Ct and the efficiency-corrected Ct model. Both models have their advantages and disadvantages in terms of simplification on the one hand and efficiency correction on the other. The particular problem of RNA integrity and its effect on relative quantification in qRT-PCR performance was tested in different bovine tissues and cell lines (n = 11). Therefore different artificial and standardized RNA degradation levels were used. Currently fully automated capillary electrophoresis systems have become the new standard in RNA quality assessment. RNA quality was rated according the RNA integrity number (RIN). Furthermore, the effect of different length of amplified products and RNA integrity on expression analyses was investigated. We found significant impact of RNA integrity on relative expression results, mainly on cycle threshold (Ct) values and a minor effect on PCR efficiency. To minimize the interference of RNA integrity on relative quantification models, we can recommend to normalize gene expression by an internal reference gene and to perform an efficiency correction. Results demonstrate that innovative new quantification methods and normalization models can improve future mRNA quantification.     

Quantitative PCR in the diagnosis of Leishmania

Polymerase chain reaction (PCR) is a sensitive and rapid method for the diagnosis of canine Leishmania infection and can be performed on a variety of biological samples, including peripheral blood, lymph node, bone marrow and skin. Standard PCR requires electrophoretic analysis of the amplification products and is usually not suitable for quantification of the template DNA (unless competitor-based or other methods are developed), being of reduced usefulness when accurate monitoring of target DNA is required. Quantitative real-time PCR allows the continuous monitoring of the accumulation of PCR products during the amplification reaction. This allows the identification of the cycle of near-logarithmic PCR product generation (threshold cycle) and, by inference, the relative quantification of the template DNA present at the start of the reaction. Since the amplification product are monitored in "real-time" as they form cycle-by-cycle, no post-amplification handling is required. The absolute quantification is performed according either to an internal standard co-amplified with the sample DNA, or to an external standard curve obtained by parallel amplification of serial known concentrations of a reference DNA sequence. From the quantification of the template DNA, an estimation of the relative load of parasites in the different samples can be obtained. The advantages compared to standard and semi-quantitative PCR techniques are reduction of the assay's time and contamination risks, and improved sensitivity. As for standard PCR, the minimal components of the quantitative PCR reaction mixture are the DNA target of the amplification, an oligonucleotide primer pair flanking the target sequence, a suitable DNA polymerase, deoxynucleotides, buffer and salts. Different technologies have been set up for the monitoring of amplification products, generally based on the use of fluorescent probes. For instance, SYBR Green technology is a non-specific detection system based on a fluorescent dsDNA intercalator and it is applicable to all potential targets. TaqMan technology is more specific since performs the direct assessment of the amount of amplified DNA using a fluorescent probe specific for the target sequence flanked by the primer pair. This probe is an oligonucleotide labelled with a reporter dye (fluorescent) and a quencher (which absorbs the fluorescent signal generated by the reporter). The thermic protocol of amplification allows the binding of the fluorescent probe to the target sequence before the binding of the primers and the starting of the polymerization by Taq polymerase. During polymerization, 5'-3' exonuclease activity of Taq polymerase digests the probe and in this way the reporter dye is released from the probe and a fluorescent signal is detected. The intensity of the signal accumulates at the end of each cycle and is related to the amount of the amplification product. In recent years, quantitative PCR methods based either on SYBR Green or TaqMan technology have been set up for the quantification of Leishmania in mouse liver, mouse skin and human peripheral blood, targeting either single-copy chromosomal or multi-copy minicircle sequences with high sensitivity and reproducibility. In particular, real-time PCR seems to be a reliable, rapid and noninvasive method for the diagnosis and follow up of visceral leishmaniasis in humans. At present, the application of real-time PCR for research and clinical diagnosis of Leishmania infection in dogs is still foreseable. As for standard PCR, the high sensitivity of real-time PCR could allow the use of blood sampling that is less invasive and easily performed for monitoring the status of the dogs. The development of a real-time PCR assay for Leishmania infantum infection in dogs could support the standard and optimized serological and PCR methods currenly in use for the diagnosis and follow-up of canine leishmaniasis, and perhaps prediction of recurrences associated with tissue loads of residual pathogens after treatment. At this regard, a TaqMan Real Time PCR method developed for the quantification of Leishmania infantum minicircle DNA in peripheral blood of naturally infected dogs sampled before and at different time points after the beginning of a standard antileishmanial therapy will be illustrated.     

Novel quantitative real-time LCR for the sensitive detection of SNP frequencies in pooled DNA: method development, evaluation and application

Background

Single nucleotide polymorphisms (SNP) have proven to be powerful genetic markers for genetic applications in medicine, life science and agriculture. A variety of methods exist for SNP detection but few can quantify SNP frequencies when the mutated DNA molecules correspond to a small fraction of the wild-type DNA. Furthermore, there is no generally accepted gold standard for SNP quantification, and, in general, currently applied methods give inconsistent results in selected cohorts. In the present study we sought to develop a novel method for accurate detection and quantification of SNP in DNA pooled samples.

Methods

The development and evaluation of a novel Ligase Chain Reaction (LCR) protocol that uses a DNA-specific fluorescent dye to allow quantitative real-time analysis is described. Different reaction components and thermocycling parameters affecting the efficiency and specificity of LCR were examined. Several protocols, including gap-LCR modifications, were evaluated using plasmid standard and genomic DNA pools. A protocol of choice was identified and applied for the quantification of a polymorphism at codon 136 of the ovine PRNP gene that is associated with susceptibility to a transmissible spongiform encephalopathy in sheep.

Conclusions

The real-time LCR protocol developed in the present study showed high sensitivity, accuracy, reproducibility and a wide dynamic range of SNP quantification in different DNA pools. The limits of detection and quantification of SNP frequencies were 0.085% and 0.35%, respectively.

Significance

The proposed real-time LCR protocol is applicable when sensitive detection and accurate quantification of low copy number mutations in DNA pools is needed. Examples include oncogenes and tumour suppressor genes, infectious diseases, pathogenic bacteria, fungal species, viral mutants, drug resistance resulting from point mutations, and genetically modified organisms in food.