Molecular quantification of environmental DNA using microfluidics and digital PCR

Real-time PCR has been widely used to evaluate gene abundance in natural microbial habitats. However, PCR-inhibitory substances often reduce the efficiency of PCR, leading to the underestimation of target gene copy numbers. Digital PCR using microfluidics is a new approach that allows absolute quantification of DNA molecules. In this study, digital PCR was applied to environmental samples, and the effect of PCR inhibitors on DNA quantification was tested. In the control experiment using λ DNA and humic acids, underestimation of λ DNA at 1/4400 of the theoretical value was observed with 6.58ngμL(-1) humic acids. In contrast, digital PCR provided accurate quantification data with a concentration of humic acids up to 9.34ngμL(-1). The inhibitory effect of paddy field soil extract on quantification of the archaeal 16S rRNA gene was also tested. By diluting the DNA extract, quantified copy numbers from real-time PCR and digital PCR became similar, indicating that dilution was a useful way to remedy PCR inhibition. The dilution strategy was, however, not applicable to all natural environmental samples. For example, when marine subsurface sediment samples were tested the copy number of archaeal 16S rRNA genes was 1.04×10(3)copies/g-sediment by digital PCR, whereas real-time PCR only resulted in 4.64×10(2)copies/g-sediment, which was most likely due to an inhibitory effect. The data from this study demonstrated that inhibitory substances had little effect on DNA quantification using microfluidics and digital PCR, and showed the great advantages of digital PCR in accurate quantifications of DNA extracted from various microbial habitats.     

Development of a real-time PCR method for quantification of the three genera Dehalobacter, Dehalococcoides, and Desulfitobacterium in microbial communities

We developed standard curves based on plasmids containing a 16S rRNA gene of a member of one of the three genera Dehalobacter, Desulfitobacterium, and Dehalococcoides. A large difference in amplification efficiency between the standard curves was observed ranging from 1.5 to 2.0. The total eubacterial 16S rRNA gene copy number determined in a sample DNA by using eubacterial primers and the three standard curves led to differences in the estimated copy numbers of a factor up to 73. However, the amplification efficiencies for one specific standard curve were the same independent of the PCR primer pair used. This allowed the determination of the abundance of a population expressed as fractional number, hence, the percentage of genus-specific copy numbers within the total eubacterial 16S rRNA gene copy numbers. Determination of the fractional numbers in DNA mixtures of known composition showed the accuracy of this approach. The average difference in threshold value between two 10-fold dilutions of DNA of pure cultures, mixtures thereof and of environmental samples was -3.45+/-0.34, corresponding to an average almost optimal amplification efficiency of 1.95. This indicated that the low amplification efficiency of certain standard curves seemed to be mainly a problem of the plasmid DNA used and not of the 16S rRNA gene of the target genera.     

Real-Time Relative qPCR without Reference to Control Samples and Estimation of Run-Specific PCR Parameters from Run-Internal Mini-Standard Curves

Background

Real-Time quantitative PCR is an important tool in research and clinical settings. Here, we describe two new approaches that broaden the scope of real-time quantitative PCR; namely, run-internal mini standard curves (RIMS) and direct real-time relative quantitative PCR (drqPCR). RIMS are an efficient alternative to traditional standard curves and provide both run-specific and target-specific estimates of PCR parameters. The drqPCR enables direct estimation of target ratios without reference to conventional control samples.

Methodology/Principal Findings

In this study, we compared RIMS-based drqPCR with classical quantifications based on external standard curves and the “comparative Ct method”. Specifically, we used a raw real-time PCR dataset as the basis for more than two-and-a-half million simulated quantifications with various user-defined conditions. Compared with classical approaches, we found that RIMS-based drqPCR provided superior precision and comparable accuracy.

Conclusions/Significance

The obviation of referencing to control samples is attractive whenever unpaired samples are quantified. This may be in clinical and research settings; for instance, studies on chimerism, TREC quantifications, copy number variations etc. Also, lab-to-lab comparability can be greatly simplified.     

Quantification of microbial communities in near-surface and deeply buried marine sediments on the Peru continental margin using real-time PCR

Deeply buried marine sediments harbour a large fraction of all prokaryotes on Earth but it is still unknown which phylogenetic and physiological microbial groups dominate the deep biosphere. In this study real-time PCR allowed a comparative quantitative microbial community analysis in near-surface and deeply buried marine sediments from the Peru continental margin. The 16S rRNA gene copy numbers of prokaryotes and Bacteria were almost identical with a maximum of 10(8)-10(10) copies cm(-3) in the near-surface sediments. Archaea exhibited one to three orders of magnitude lower 16S rRNA gene copy numbers. The 18S rRNA gene of Eukarya was always at least three orders of magnitude less abundant than the 16S rRNA gene of prokaryotes. The 16S rRNA gene of the Fe(III)- and Mn(IV)-reducing bacterial family Geobacteraceae and the dissimilatory (bi)sulfite reductase gene (dsrA) of sulfate-reducing prokaryotes were abundant with 10(6)-10(8) copies cm(-3) in near-surface sediments but showed lower numbers and an irregular distribution in the deep sediments. The copy numbers of all genes decreased with sediment depth exponentially. The depth gradients were steeper for the gene copy numbers than for numbers of total prokaryotes (acridine orange direct counts), which reflects the ongoing degradation of the high-molecular-weight DNA with sediment age and depth. The occurrence of eukaryotic DNA also suggests DNA preservation in the deeply buried sediments.     

Detection of bacterial pathogens in municipal wastewater using an oligonucleotide microarray and real-time quantitative PCR

As a first step toward building a comprehensive microarray, two low density DNA microarrays were constructed and evaluated for the accurate detection of wastewater pathogens. The first one involved the direct hybridization of wastewater microbial genomic DNA to the functional gene probes while the second involved PCR amplification of 23S ribosomal DNA. The genomic DNA microarray employed 10 functional genes as detection targets. Sensitivity of the microarray was determined to be approximately 1.0 microg of Esherichia coli genomic DNA, or 2 x 10(8) copies of the target gene, and only E. coli DNA was detected with the microarray assay using municipal raw sewage. Sensitivity of the microarray was enhanced approximately by 6 orders of magnitude when the target 23S rRNA gene sequences were PCR amplified with a novel universal primer set and allowed hybridization to 24 species-specific oligonucleotide probes. The minimum detection limit was estimated to be about 100 fg of E. coli genomic DNA or 1.4 x 10(2) copies of the 23S rRNA gene. The PCR amplified DNA microarray successfully detected multiple bacterial pathogens in wastewater. As a parallel study to verify efficiency of the DNA microarray, a real-time quantitative PCR assay was also developed based on the fluorescent TaqMan probes (Applied Biosystems).     

Polymerase chain reaction amplification length-dependent ethidium monoazide suppression power for heat-killed cells of Enterobacteriaceae

The polymerase chain reaction (PCR) can confirm the presence of bacteria, but it is unable to differentiate between live and dead bacteria. Although ethidium monoazide (EMA)- and propidium monoazide (PMA)-based PCR have been evaluated, a quantity of ≥ 10(3)cells/ml dead cells produces a false-positive reading at 40 to 50 cycles (K. Rudi et al., Appl. Environ. Microbiol. 71 (2005) 1018-1024). After confirming the precision of real-time PCR of a long DNA target (16S or 23S ribosomal RNA [rRNA] gene, 1490 or 2840 bp), we evaluated the degree of suppression of an EMA treatment on the 16S/23S PCR using various amplification lengths (110-2840 bp) with heat-killed cells of Enterobacteriaceae (e.g., Salmonella enteritidis). We found that the inhibition rate was proportional to the PCR amplification length; short DNA (110 bp) amplification slightly delayed the threshold cycle (C(T)) of heat-killed cells of Enterobacteriaceae when compared with no EMA treatment. Regardless of the amplification length, the C(T) delay using live cells of Enterobacteriaceae with EMA was negligible. Thus, our real-time PCR of a long DNA (16S or 23S) template following EMA treatment is a rapid viable bacterial assay, which can potentially target all genera, for testing pasteurized milk that may have originally been contaminated with high levels of dead bacteria.     

High-sensitivity quantitative PCR platform

Real-time PCR methods have become widely used within the past few years. However, real-time PCR is rarely used to study chronic diseases with low pathogen loads, presumably because of insufficient sensitivity. In this report, we developed an integrated nucleic acid isolation and real-time PCR platform that vastly improved the sensitivity of the quantitative detection of the intracellular bacterium, Chlamydia spp., by fluorescence resonance energy transfer real-time PCR. Determinants of the overall detection sensitivity were analyzed by extracting nucleic acids from bovine milk specimens spiked with low amounts of chlamydial organisms. Nucleic acids were optimally preserved and recovered by collection in guanidinium stabilization buffer, binding to a matrix of glass fiber fleece, and elution in low volume. Step-down thermal cycling and an excess of hot-start Taq polymerase vastly improved the robustness and sensitivity of the real-time PCR while essentially maintaining 100% specificity. The amplification of Chlamydia 23S rRNA allowed for the differentiation of chlamydial species and was more robust at low target numbers than amplification of the omp1 gene. The best combined method detected single targets per a 100-microL specimen equivalent in a 5-microL real-time PCR input. In an initial application, this high-sensitivity real-time PCR platform demonstrated a high prevalence of chlamydial infection in cattle.     

A proportional analysis method using non-kinetic real-time PCR

Prompted by increasing interest in proportional analysis of genetic types, we developed a simple assay technique for determining the ratio of a specific target gene in the total genes that can be amplified with the same PCR primer. The key feature of this method is that the following two tasks are performed in a single-tube real-time PCR system: task 1, PCR amplification of the total genes including the target using a labeled PCR primer, with concurrent monitoring of the total copy number of the PCR product; task 2, detection of the signal of the target gene at each cycle of amplification, using a labeled nucleotide probe. In principle, the ratio of the target gene to the total genes is represented by the signal detected in 'task 2' at the cycle in which the PCR product reached a prescribed copy number (assessed by 'task 1').     

Quantification of the c-myc gene in gastric carcinomas by the triplex polymerase chain reaction and high performance liquid chromatography.

The triplex polymerase chain reaction (PCR) and high performance liquid chromatography (HPLC) techniques were used to examine the state of amplification of the c-myc gene in gastric carcinomas. Sequences from the c-myc gene and from the two control genes were coamplified by PCR. The coamplified PCR products were separated and quantified by HPLC and the copy numbers of the c-myc gene were calculated by comparing the peak areas generated by PCR products. Increased copy numbers of the c-myc gene were found in 2 of 5 patients.     

Real-time PCR determination of rRNA gene copy number: absolute and relative quantification assays with <Emphasis Type="Italic">Escherichia coli</Emphasis>

Real-time polymerase chain reaction (PCR)-based methodology for the determination of rRNA gene (rrn) copy number was introduced and demonstrated. Both absolute and relative quantifications were tested with Escherichia coli. The separate detection of rRNA gene and chromosomal DNA was achieved using two primer sets, specific for 16S rRNA gene and for D-1-deoxyxylulose 5-phosphate synthase gene (dxs), respectively. As dxs is a single-copy gene of E. coli chromosomal DNA, the rrn copy number can be determined as the copy ratio of rrn to dxs. This methodology was successfully applied to determine the rrn copy number in E. coli cells. The results from absolute and relative quantifications were identical and highly reproducible with coefficient of variation (CV) values of 1.8–4.6%. The estimated rrn copy numbers also corresponded to the previously reported value in E. coli (i.e., 7), indicating that the results were reliable. The methodology introduced in this study is faster and cost-effective without safety problems compared to the traditionally used Southern blot analysis. The fundamentals in our methodology would be applicable to any microorganism, as long as having the sequence information of the rRNA gene and another chromosomal gene with a known copy number.     

Theoretical uncertainty of measurements using quantitative polymerase chain reaction.

Current quantitative polymerase chain reaction (PCR) protocols are only indicative of the quantity of a target sequence relative to a standard, because no means of estimating the amplification rate is yet available. The variability of PCR performed on isolated cells has already been reported by several authors, but it could not be extensively studied, because of lack of a system for doing kinetic data acquisition and of statistical methods suitable for analyzing this type of data. We used the branching process theory to simulate and analyze quantitative kinetic PCR data. We computed the probability distribution of the offspring of a single molecule. We demonstrated that the rate of amplication has a severe influence on the shape of this distribution. For high values of the amplification rate, the distribution has several maxima of probability. A single amplification trajectory is used to estimate the initial copy number of the target sequence as well as its confidence interval, provided that the amplification is done over more than 20 cycles. The consequence of possible molecular fluctuations in the early stage of amplification is that small copy numbers result in relatively larger intervals than large initial copy numbers. The confidence interval amplitude is the theoretical uncertainty of measurements using quantitative PCR. We expect these results to be applicable to the data produced by the next generation of thermocyclers for quantitative applications.     

Quantitative Detection of the nosZ Gene, Encoding Nitrous Oxide Reductase, and Comparison of the Abundances of 16S rRNA, narG, nirK, and nosZ Genes in Soils

Nitrous oxide (N₂O) is an important greenhouse gas in the troposphere controlling ozone concentration in the stratosphere through nitric oxide production. In order to quantify bacteria capable of N₂O reduction, we developed a SYBR green quantitative real-time PCR assay targeting the nosZ gene encoding the catalytic subunit of the nitrous oxide reductase. Two independent sets of nosZ primers flanking the nosZ fragment previously used in diversity studies were designed and tested (K. Kloos, A. Mergel, C. Rösch, and H. Bothe, Aust. J. Plant Physiol. 28:991-998, 2001). The utility of these real-time PCR assays was demonstrated by quantifying the nosZ gene present in six different soils. Detection limits were between 10¹ and 10² target molecules per reaction for all assays. Sequence analysis of 128 cloned quantitative PCR products confirmed the specificity of the designed primers. The abundance of nosZ genes ranged from 10⁵ to 10⁷ target copies g⁻¹ of dry soil, whereas genes for 16S rRNA were found at 10⁸ to 10⁹ target copies g⁻¹ of dry soil. The abundance of narG and nirK genes was within the upper and lower limits of the 16S rRNA and nosZ gene copy numbers. The two sets of nosZ primers gave similar gene copy numbers for all tested soils. The maximum abundance of nosZ and nirK relative to 16S rRNA was 5 to 6%, confirming the low proportion of denitrifiers to total bacteria in soils.     

Quantitative detection of the free-living amoeba Hartmannella vermiformis in surface water by using real-time PCR

A real-time PCR-based method targeting the 18S rRNA gene was developed for the quantitative detection of Hartmannella vermiformis, a free-living amoeba which is a potential host for Legionella pneumophila in warm water systems and cooling towers. The detection specificity was validated using genomic DNA of the closely related amoeba Hartmannella abertawensis as a negative control and sequence analysis of amplified products from environmental samples. Real-time PCR detection of serially diluted DNA extracted from H. vermiformis was linear for microscopic cell counts between 1.14 x 10(-1) and 1.14 x 10(4) cells per PCR. The genome of H. vermiformis harbors multiple copies of the 18S rRNA gene, and an average number (with standard error) of 1,330 +/- 127 copies per cell was derived from real-time PCR calibration curves for cell suspensions and plasmid DNA. No significant differences were observed between the 18S rRNA gene copy numbers for trophozoites and cysts of strain ATCC 50237 or between the copy numbers for this strain and strain KWR-1. The developed method was applied to water samples (200 ml) collected from a variety of lakes and rivers serving as sources for drinking water production in The Netherlands. Detectable populations were found in 21 of the 28 samples, with concentrations ranging from 5 to 75 cells/liter. A high degree of similarity (> or =98%) was observed between sequences of clones originating from the different surface waters and between these clones and the reference strains. Hence, H. vermiformis, which is highly similar to strains serving as hosts for L. pneumophila, is a common component of the microbial community in fresh surface water.     

同步PCR技术及其在植物核酸分子定量中的应用

同步PCR是一种集生化、光电和计算机技术于一体的封闭式DNA扩增系统,采用荧光染料将扩增与检测过程结合在一起,实现了在PCR过程中在线显示PCR反应,通过检测荧光强度来绝对定量起始模板的拷贝数.该技术大大简化和加速了核酸分子的定量过程,不仅快速、灵敏、准确、重复性好,而且很容易计算出待测样品中核酸分子的绝对起始拷贝数.同微阵列等分子生物技术一起,同步PCR技术将会在功能基因解析和病害分子诊断等方面发挥重要作用.本综述除了介绍同步PCR技术的原理和应用外,还介绍了定量拟南芥Aux/IAA基因的转录水平的实验,并就同步PCR操作过程中的问题进行了讨论.     

Estimating number of transgene copies in transgenic rapeseed by real-time PCR assay with<Emphasis Type="Italic">HMG I/Y</Emphasis> as an endogenous reference gene

In transgenic plants, the number of transgene copies can greatly influence the level of expression and genetic stability of the target gene. Transgene copy numbers are estimated by Southern blot analysis, which is laborious and time-consuming, requires relatively large amounts of plant materials, and may involve hazardous radioisotopes. Here we report the development of a sensitive, convenient real-time PCR technique for estimating the number of transgene copies in transgenic rapeseed. This system uses TaqMan quantitative real-time PCR and comparison with a novel, confirmed single-copy endogenous reference gene, high-mobile-group protein I/Y (HMG I/Y), to determine the numbers of copies of exogenous β-glucuronidase (GUS) and neomycin phosphotransferase II (nptII) genes. TheGUS andnptII copy numbers in primary transformants (T₀) were calculated by comparing threshold cycle (C _T) values of theGUS andnptII genes with those of the internal standard,HMG I/Y. This method is more convenient and accurate than Southern blotting because the number of copies of the exogenous gene could be directly deduced by comparing itsC _T value to that of the single-copy endogenous gene in each sample. Unlike other similar procedures of real-time PCR assay, this method does not require identical amplification efficiencies between the PCR systems for target gene and endogenous reference gene, which can avoid the bias that may result from slight variations in amplification efficiencies between PCR systems of the target and endogenous reference genes.     

PCR-Induced Sequence Artifacts and Bias: Insights from Comparison of Two 16S rRNA Clone Libraries Constructed from the Same Sample

The contribution of PCR artifacts to 16S rRNA gene sequence diversity from a complex bacterioplankton sample was estimated. Taq DNA polymerase errors were found to be the dominant sequence artifact but could be constrained by clustering the sequences into 99% sequence similarity groups. Other artifacts (chimeras and heteroduplex molecules) were significantly reduced by employing modified amplification protocols. Surprisingly, no skew in sequence types was detected in the two libraries constructed from PCR products amplified for different numbers of cycles. Recommendations for modification of amplification protocols and for reporting diversity estimates at 99% sequence similarity as a standard are given.     

Roles of the 2 microns gene products in stable maintenance of the 2 microns plasmid of Saccharomyces cerevisiae.

We have examined the replication and segregation of the Saccharomyces cerevisiae 2 microns circle. The amplification of the plasmid at low copy numbers requires site-specific recombination between the 2 microns inverted repeat sequences catalyzed by the plasmid-encoded FLP gene. No other 2 microns gene products are required. The overexpression of FLP in a strain carrying endogenous 2 microns leads to uncontrolled plasmid replication, longer cell cycles, and cell death. Two different assays show that the level of Flp activity decreases with increasing 2 microns copy number. This regulation requires the products of the REP1 and REP2 genes. These gene products also act together to ensure that 2 microns molecules are randomly segregated between mother and daughter cells at cell division.     

Theoretical and experimental dissection of competitive PCR for accurate quantification of DNA

We frequently use competitive PCR in the plateau phase in quantifying DNA species with a small number of cells. However, the basic issues of this method are poorly understood. Here, first we analyze this method theoretically under a generalized condition that competitor and target DNA products accumulate with different amplification efficiencies. We show a theoretical reason that competitive PCR might quantify DNA more accurately during the plateau phase than during the exponential phase. Second, we demonstrate that the theoretical predictions are supported by the experimental results of beta-globin gene amplification using the lysates of human diploid fibroblast WS1 cells. We also demonstrate that we can correctly quantify target DNA by keeping the starting concentration of target DNA close to a constant preset value while using a constant number of PCR cycles and by using WS1 cells as control. Finally, we show the experimental errors in routine measurements of c-myc copy number/cell in human leukemia HL-60 cells with various levels of c-myc multiplication. The number of c-myc copies/cell was determined with an error rate of less than 10%, where agarose gel bands were stained with ethidium bromide for the product quantitation.     

Determination of Transgene Copy Number in Stably Transfected Mammalian Cells by PCR-Capillary Electrophoresis Assay

The quantitative determination of transgene copy number in stably transfected mammalian cells has been traditionally estimated by Southern blot analysis. Recently, other methods have become available for appraisal of gene copy number, such as real-time PCR. Herein we describe a new method based on a fluorescently labeled PCR, followed by capillary electrophoresis. We amplified our target gene (prothrombin) and the internal control originating from genomic DNA (18S rRNA) in the same PCR tube and calculated the mean peak height ratio of the target:control gene for every cell clone sample. With this approach we identified stably transfected cell clones bearing the same transgene copy number. The results of our assay were confirmed by real-time PCR. Our method proves to be fast, low-cost, and reproducible compared with traditionally used methods. This assay can be used as a rapid screening tool for the determination of gene copy number in gene expression experiments.