MPN estimation of qPCR target sequence recoveries from whole cell calibrator samples

DNA extracts from enumerated target organism cells (calibrator samples) have been used for estimating Enterococcus cell equivalent densities in surface waters by a comparative cycle threshold (Ct) qPCR analysis method. To compare surface water Enterococcus density estimates from different studies by this approach, either a consistent source of calibrator cells must be used or the estimates must account for any differences in target sequence recoveries from different sources of calibrator cells. In this report we describe two methods for estimating target sequence recoveries from whole cell calibrator samples based on qPCR analyses of their serially diluted DNA extracts and most probable number (MPN) calculation. The first method employed a traditional MPN calculation approach. The second method employed a Bayesian hierarchical statistical modeling approach and a Monte Carlo Markov Chain (MCMC) simulation method to account for the uncertainty in these estimates associated with different individual samples of the cell preparations, different dilutions of the DNA extracts and different qPCR analytical runs. The two methods were applied to estimate mean target sequence recoveries per cell from two different lots of a commercially available source of enumerated Enterococcus cell preparations. The mean target sequence recovery estimates (and standard errors) per cell from Lot A and B cell preparations by the Bayesian method were 22.73 (3.4) and 11.76 (2.4), respectively, when the data were adjusted for potential false positive results. Means were similar for the traditional MPN approach which cannot comparably assess uncertainty in the estimates. Cell numbers and estimates of recoverable target sequences in calibrator samples prepared from the two cell sources were also used to estimate cell equivalent and target sequence quantities recovered from surface water samples in a comparative Ct method. Our results illustrate the utility of the Bayesian method in accounting for uncertainty, the high degree of precision attainable by the MPN approach and the need to account for the differences in target sequence recoveries from different calibrator sample cell sources when they are used in the comparative Ct method.     

Incorporation of measurement of DNA integrity into qPCR assays

Optimal accuracy of quantitative PCR (qPCR) requires correction for integrity of the target sequence. Here we combine the mathematics of the Poisson distribution and exponential amplification to show that the frequency of lesions per base (which prevent PCR amplification) can be derived from the slope of the regression line between cycle threshold (Ct) and amplicon length. We found that the amplifiable fraction (AF) of a target can be determined from this frequency and the target length. Experimental results from this method correlated with both the magnitude of a damaging agent and with other measures of DNA damage. Applying the method to a reference sequence, we determined the values for lesions/base in control samples, as well as in the AFs of the target sequence in qPCR samples collected from leukemic patients. The AFs used to calculate the final qPCR result were generally >0.5, but were <0.2 in a few samples, indicating significant degradation. We conclude that DNA damage is not always predictable; quantifying the DNA integrity of a sample and determining the AF of a specific qPCR target will improve the accuracy of qPCR and aid in the interpretation of negative results.     

Analysis of qPCR data by converting exponentially related Ct values into linearly related X0 values

A common method for calculating results from qPCR experiments is the comparative Ct method, also called the 2(-ΔΔCt) method. However, several assumptions are included in the 2(-ΔΔCt) method and standard statistical analyses are not directly applicable. Here, we describe a different method, the X(0) method, for result calculations and statistical analysis from qPCR experiments. The X(0) method differs from the 2(-ΔΔCt) method by introducing a conversion of the exponentially related Ct values into linearly related X(0) values, which represent the amount of starting material in a qPCR experiment. Results calculated by the X(0) method are illustrated for qPCR experiments with technical and biological replicates, including procedures to calculate standard deviations. Incorporation of primer efficiencies in calculations by the X(0) method is also described. Altogether, the X(0) method constitutes a very simple and accurate alternative to the 2(-ΔΔCt) method for result calculations from qPCR data.     

Effectiveness of qPCR permutations, internal controls and dilution as means for minimizing the impact of inhibition while measuring Enterococcus in environmental waters

Aims: Draft criteria for the optional use of qPCR for recreational water quality monitoring have been published in the United States. One concern is that inhibition of the qPCR assay can lead to false‐negative results and potentially inadequate public health protection. We evaluate the effectiveness of strategies for minimizing the impact of inhibition. Methods and Results: Five qPCR method permutations for measuring Enterococcus were challenged with 133 potentially inhibitory fresh and marine water samples. Serial dilutions were conducted to assess Enterococcus target assay inhibition, to which inhibition identified using four internal controls (IC) was compared. The frequency and magnitude of inhibition varied considerably among qPCR methods, with the permutation using an environmental master mix performing substantially better. Fivefold dilution was also effective at reducing inhibition in most samples (>78%). ICs were variable and somewhat ineffective, with 54–85% agreement between ICs and serial dilution. Conclusions: The current IC methods appear to not accurately predict Enterococcus inhibition and should be used with caution; fivefold dilution and the use of reagents designed for environmental sample analysis (i.e. more robust qPCR chemistry) may be preferable. Significance and Impact of the Study: Suitable approaches for defining, detecting and reducing inhibition will improve implementation of qPCR for water monitoring.     

Quantitative PCR Method for Enumeration of Cells of Cryptic Species of the Toxic Marine Dinoflagellate Ostreopsis spp. in Coastal Waters of Japan

Monitoring of harmful algal bloom (HAB) species in coastal waters is important for assessment of environmental impacts associated with HABs. Co-occurrence of multiple cryptic species such as toxic dinoflagellate Ostreopsis species make reliable microscopic identification difficult, so the employment of molecular tools is often necessary. Here we developed new qPCR method by which cells of cryptic species can be enumerated based on actual gene number of target species. The qPCR assay targets the LSU rDNA of Ostreopsis spp. from Japan. First, we constructed standard curves with a linearized plasmid containing the target rDNA. We then determined the number of rDNA copies per cell of target species from a single cell isolated from environmental samples using the qPCR assay. Differences in the DNA recovery efficiency was calculated by adding exogenous plasmid to a portion of the sample lysate before and after DNA extraction followed by qPCR. Then, the number of cells of each species was calculated by division of the total number of rDNA copies of each species in the samples by the number of rDNA copies per cell. To test our procedure, we determined the total number of rDNA copies using environmental samples containing no target cells but spiked with cultured cells of several species of Ostreopsis. The numbers estimated by the qPCR method closely approximated total numbers of cells added. Finally, the numbers of cells of target species in environmental samples containing cryptic species were enumerated by the qPCR method and the total numbers also closely approximated the microscopy cell counts. We developed a qPCR method that provides accurate enumeration of each cryptic species in environments. This method is expected to be a powerful tool for monitoring the various HAB species that occur as cryptic species in coastal waters.     

Evaluation of a Rapid, Quantitative Real-Time PCR Method for Enumeration of Pathogenic Candida Cells in Water

Quantitative PCR (QPCR) technology, incorporating fluorigenic 5′ nuclease (TaqMan) chemistry, was utilized for the specific detection and quantification of six pathogenic species of Candida (C. albicans, C. tropicalis, C. krusei, C. parapsilosis, C. glabrata and C. lusitaniae) in water. Known numbers of target cells were added to distilled and tap water samples, filtered, and disrupted directly on the membranes for recovery of DNA for QPCR analysis. The assay's sensitivities were between one and three cells per filter. The accuracy of the cell estimates was between 50 and 200% of their true value (95% confidence level). In similar tests with surface water samples, the presence of PCR inhibitory compounds necessitated further purification and/or dilution of the DNA extracts, with resultant reductions in sensitivity but generally not in quantitative accuracy. Analyses of a series of freshwater samples collected from a recreational beach showed positive correlations between the QPCR results and colony counts of the corresponding target species. Positive correlations were also seen between the cell quantities of the target Candida species detected in these analyses and colony counts of Enterococcus organisms. With a combined sample processing and analysis time of less than 4 h, this method shows great promise as a tool for rapidly assessing potential exposures to waterborne pathogenic Candida species from drinking and recreational waters and may have applications in the detection of fecal pollution.     

Bayesian estimation of ancestral character states on phylogenies

Biologists frequently attempt to infer the character states at ancestral nodes of a phylogeny from the distribution of traits observed in contemporary organisms. Because phylogenies are normally inferences from data, it is desirable to account for the uncertainty in estimates of the tree and its branch lengths when making inferences about ancestral states or other comparative parameters. Here we present a general Bayesian approach for testing comparative hypotheses across statistically justified samples of phylogenies, focusing on the specific issue of reconstructing ancestral states. The method uses Markov chain Monte Carlo techniques for sampling phylogenetic trees and for investigating the parameters of a statistical model of trait evolution. We describe how to combine information about the uncertainty of the phylogeny with uncertainty in the estimate of the ancestral state. Our approach does not constrain the sample of trees only to those that contain the ancestral node or nodes of interest, and we show how to reconstruct ancestral states of uncertain nodes using a most-recent-common-ancestor approach. We illustrate the methods with data on ribonuclease evolution in the Artiodactyla. Software implementing the methods (BayesMultiState) is available from the authors.     

Improved real-time PCR method for quantification of the abundance of all known ribotypes of the ichthyotoxic dinoflagellate Cochlodinium polykrikoides by comparing 4 different preparation methods

Red tides by the ichthyotoxic dinoflagellate Cochlodinium polykrikoides have caused large scaled mortality of fish and great loss in aquaculture industry in many countries. Detecting and quantifying the abundance of this species are the most critical step in minimizing the loss. The conventional quantitative real-time PCR (qPCR) method has been used for quantifying the abundance of this species. However, when analyzing > 500 samples collected during huge C. polykrikoides red tides in South Sea of Korea in 2014, this conventional method and the previously developed specific primer and probe set for C. polykrikoides did not give reasonable abundances when compared with cell counting data. Thus improved qPCR methods and a new specific primer and probe set reflecting recent discovery of 2 new ribotypes have to be developed. A new species-specific primer and probe set for detecting all 3 ribotypes of C. polykrikoides was developed and provided in this study. Furthermore, because the standard curve between cell abundance and threshold cycle value (Ct) is critical, the efficiencies of 4 different preparation methods used to determine standard curves were comparatively evaluated. The standard curves were determined by using the following 4 different preparations: (1) extraction of DNA from a dense culture of C. polykrikoides followed by serial dilution of the extracted DNA (CDD method), (2) extraction of DNA from each of the serially diluted cultures with different concentrations of C. polykrikoides cultures (CCD method), (3) extraction of DNA from a dense field sample of C. polykrikoides collected from natural seawater and then dilution of the extracted DNA in serial (FDD method), and (4) extraction of DNA from each of the serially diluted field samples having different concentrations of C. polykrikoides (FCD method). These 4 methods yielded different results. The abundances of C. polykrikoides in the samples collected from the coastal waters of South Sea, Korea, in 2014–2015, obtained using the standard curves determined by the CCD and the FCD methods, were the most similar (0.93–1.03 times) and the second closest (1.16–1.33 times) to the actual cell abundances obtained by enumeration of cells. Thus, our results suggest that the CCD method is a more effective tool to quantify the abundance of C. polykrikoides than the conventional method, CDD, and the FDD and FCD methods.     

A new immunoprecipitation-real time quantitative PCR assay for anti-Th/To and anti-U3RNP antibody detection in systemic sclerosis

Introduction

Classic anti-nucleolar antibodies anti-Th/To and U3 ribonucleoprotein (-U3RNP) can help in the diagnosis, prediction of organ involvement and prognosis in systemic sclerosis (SSc); however, no validated commercial assay is available. We aimed at establishing a novel quantitative real time PCR (qPCR) method to detect these antibodies.

Methods

Standard immunoprecipitation (IP) was performed using K562 cell extract and RNA components were extracted. cDNA was reverse transcribed from RNA components and Th RNA and U3 RNA were detected by qPCR using custom primers. Cycle threshold (Ct) values were compared in a titration experiment to determine the assay efficacy. The new assay was evaluated by testing 22 anti-Th/To and 12 anti-U3RNP positive samples in addition to 88 controls, and the results were compared with IP as a gold standard.

Results

By testing serial 1:8 dilutions of cell lysate as the substrate in the IP step, RNA extracted after IP, and its derived cDNA, linear dose response curves were noted for both anti-Th/To and -U3RNP. With every dilution, Ct values changed approximately three as expected, reflecting the eight-fold difference of cDNA. The Ct difference between positive and negative samples was 8 to 13, which was similar throughout the dilutions. In the specificity analysis, the Ct values of positive samples were clearly different from the negative groups and the results by qPCR had a near perfect correlation with IP.

Conclusions

Our new method readily detects these two clinically important antibodies in SSc. Making tests for anti-Th/To and -U3RNP antibodies widely available to clinicians should be helpful in the diagnosis and follow-up of SSc patients.     

Identification of suitable reference genes for expression studies in pomegranate under different biotic and abiotic stress conditions

Pomegranate (Punica granatum L.) is an important economic fruit crop, facing many biotic and abiotic challenges during cultivation. Several research programs are in progress to understand both biotic and abiotic stress factors and mitigate these challenges using gene expression studies based on the qPCR approach. However, research publications are not available yet to select the standard reference gene for normalizing target gene expression values in pomegranate. The most suitable candidate reference gene is required to ensure precise and reliable results for qPCR analysis. Eight candidate reference genes' stability was evaluated under different stress conditions using different algorithms such as ?Ct, geNorm, BestKeeper, NormFinder, and RefFinder. The various algorithms revealed that EFA1 and 18S rRNA were common and most stable reference genes (RGs) under abiotic and wilt stress. Whereas comprehensive ranking by RefFinder showed GAPDH and CYPF were the most stable RGs under combined biotic (pooled samples of all biotic stress) and bacterial blight samples. For normalizing target gene expression under wilt, nematode, bacterial blight, and abiotic stress conditions both GAPDH and CYPFreference genes are adequate for qPCR. The above data provide comprehensive details for the selection of a candidate reference gene in various stresses in pomegranate

     

Quantification of Frankia in soils using SYBR Green based qPCR

A SYBR Green based qPCR method was developed for the quantification of clusters 1 and 3 of the actinomycete Frankia in soils. Primer nifHr158 was designed to be used as reverse primer in combination with forward primer nifHf1 specifically amplifying a 191-bp fragment of the nifH gene of these Frankia. The primer combination was tested for specificity on selected pure cultures, and by comparative sequence analyses of randomly selected clones of a clone library generated with these primers from soil DNA extracts. After adjustments of DNA extraction conditions, and the determination of extraction efficiencies used for sample normalization, copy numbers of nifH genes representing Frankia of clusters 1 and 3 were quantified in different mineral soils, resulting in cell density estimates for these Frankia of up to 10(6) cells [g soil {dry weight}](-1) depending on the soil. Despite indications that the nifH gene is not a perfect target for the quantification of Frankia, the qPCR method described here provides a new tool for the quantification and thus a more complete examination of the ecology of Frankia in soils.     

Comparison of genovars and Chlamydia trachomatis infection loads in ocular samples from children in two distinct cohorts in Sudan and Morocco

Trachoma is a blinding disease caused by repeated conjunctival infection with different Chlamydia trachomatis (Ct) genovars. Ct B genovars have been associated with more severe trachoma symptoms. Here, we investigated associations between Ct genovars and bacterial loads in ocular samples from two distinct geographical locations in Africa, which are currently unclear. We tested ocular swabs from 77 Moroccan children (28 with trachomatous inflammation-follicular (TF) and 49 healthy controls), and 96 Sudanese children (54 with TF and 42 healthy controls) with a Ct-specific real-time polymerase chain reaction (PCR) assay. To estimate bacterial loads, Ct-positive samples were further processed by multiplex real-time qPCR to amplify the chromosomal outer membrane complex B and plasmid open reading frame 2 of Ct. Genotyping was performed by PCR-based amplification of the outer membrane protein A gene (~1120 base pairs) of Ct and Sanger sequencing. Ct-positivities among the Moroccan and Sudanese patient groups were 60·7% and 31·5%, respectively. Significantly more Sudanese patients than Moroccan patients were genovar A-positive. In contrast, B genovars were significantly more prevalent in Moroccan patients than in Sudanese patients. Significantly higher Ct loads were found in samples positive for B genovars (598596) than A genovar (51005). Geographical differences contributed to the distributions of different ocular Ct genovars. B genovars may induce a higher bacterial load than A genovars in trachoma patients. Our findings emphasize the importance of conducting broader studies to elucidate if the noted difference in multiplication abilities are genovar and/or endemicity level dependent.     

Interpreting scratch assays using pair density dynamics and approximate Bayesian computation

Quantifying the impact of biochemical compounds on collective cell spreading is an essential element of drug design, with various applications including developing treatments for chronic wounds and cancer. Scratch assays are a technically simple and inexpensive method used to study collective cell spreading; however, most previous interpretations of scratch assays are qualitative and do not provide estimates of the cell diffusivity, D, or the cell proliferation rate, λ. Estimating D and λ is important for investigating the efficacy of a potential treatment and provides insight into the mechanism through which the potential treatment acts. While a few methods for estimating D and λ have been proposed, these previous methods lead to point estimates of D and λ, and provide no insight into the uncertainty in these estimates. Here, we compare various types of information that can be extracted from images of a scratch assay, and quantify D and λ using discrete computational simulations and approximate Bayesian computation. We show that it is possible to robustly recover estimates of D and λ from synthetic data, as well as a new set of experimental data. For the first time, our approach also provides a method to estimate the uncertainty in our estimates of D and λ. We anticipate that our approach can be generalized to deal with more realistic experimental scenarios in which we are interested in estimating D and λ, as well as additional relevant parameters such as the strength of cell-to-cell adhesion or the strength of cell-to-substrate adhesion.     

Combining maxRatio analysis with real-time PCR and its potential application for the prediction of Meloidogyne incognita in field samples

Diagnosing and quantifying plant-parasitic nematodes is critical for efficient nematode management. Several studies have been performed intending to demonstrate nematode quantification via real-time quantitative PCR. However, most of the studies used dilution of DNA templates to make standard curves, while few studies used samples with different nematode numbers to make the standard curve, resulting in a high standard error. The objective of the present study was to develop a high quality standard curve using samples containing different numbers of the root-knot nematode Meloidogyne incognita and evaluate the results of real time qPCR with maxRatio analysis. The results showed that a high quality standard curve was obtained with different nematode numbers using specific primers and cycle threshold (Ct)-PCR (R(2)=0.9962, P<0.001, n=9). With the maxRatio analysis, the fractional cycle number (FCN)-PCR cycle curve and adjusted FCN (FCNadj)-PCR cycle curve had similar patterns as those of the Ct-PCR cycle curve. For quantification of nematodes in field soil samples, qPCR estimations with a FCNadj-PCR cycle standard curve was very close to microscope counting of second-stage juveniles (R(2)=0.9064, P<0.001, n=10), qPCR estimations with a FCN-PCR cycle standard curve was comparably good (R(2)=0.8509, P<0.001, n=10), and the biases with a Ct-PCR cycle standard curve were large (R(2)=0.7154, P<0.001, n=10). Moreover, we found that the concentration of Triton X-100 had less of an effect on FCN as compared to Ct, with delta FCN 0.52, and delta Ct 3.94 at 0.8% Triton. The present study suggests, that combined with maxRatio methods, real time qPCR could be a practical approach for quantifying M. incognita in field samples.     

Campylobacters and bacteriophages in the surface waters of Canterbury (New Zealand)

Aim:  To determine the relationship between the presence of thermotolerant campylobacters and their bacteriophages (phages) in surface waters for the potential to use phages as an indicator of Campylobacter spp.
Methods and Results:  Thermotolerant campylobacters were enumerated in 53 water samples using a three tube most probable number (MPN) series in m-Exeter broth. The presence of phages in the same samples was tested using two approaches: qualitative enrichment with five different Campylobacter hosts and a quantitative membrane concentration method. Phages infecting an Escherichia coli O157:H7 isolate were also enumerated by the membrane concentration method. Campylobacter spp. were isolated from 45/53 (85%) of the samples at 0.4–110 MPN 100 ml⁻¹. No Campylobacter phages were isolated, but coliphages were present in 43/46 (93%) of samples.
Conclusions:  The membrane concentration method recovered >80% of Campylobacter phages from spiked samples. The absence of Campylobacter phages in environmental samples, from both enrichment and concentration methods, suggests that, if present, they are at very low titres.
Significance and Impact of the Study:  Testing for Campylobacter phages as an indicator of Campylobacter spp. presence is not effective. The quantitative data for Campylobacter spp. will be useful for risk assessment purposes.     

Correlation between quantitative PCR and culture-based methods for measuring Enterococcus spp. over various temporal scales at three California marine beaches

Several studies have examined how fecal indicator bacteria (FIB) measurements compare between quantitative PCR (qPCR) and the culture methods it is intended to replace. Here, we extend those studies by examining the stability of that relationship within a beach, as affected by time of day and seasonal variations in source. Enterococcus spp. were quantified at three southern California beaches in the morning and afternoon using two qPCR assays, membrane filtration, and defined-substrate testing. While qPCR and culture-based measurements were consistently and significantly correlated, strength of the correlation varied both among and within beaches. Correlations were higher in the morning (0.45 < ρ < 0.74 [P < 0.002]) than in the afternoon (0.18 < ρ < 0.45 [P < 0.021]) and higher when the fecal contamination was concentrated (0.38 < ρ < 0.83 [P < 0.001]) than when it was diffuse (0.19 < ρ < 0.34 [P < 0.003]). The ratios of culture-based and qPCR results (CFU or most probable number [MPN] per calibrator cell equivalents [CCE]) also varied spatially and temporally. Ratios ranged between 0.04 and 0.85 CFU or MPN per CCE and were lowest at the beach affected by diffuse pollution. Patterns in the ratios over the course of the day were dissimilar across beaches, increasing with time at one beach and decreasing at another. The spatial and temporal variability we observed indicate that the empirical relationship between culture-based and qPCR results is not universal, even within a beach.     

Quantitative PCR targeting 16S rRNA and reductive dehalogenase genes simultaneously monitors multiple Dehalococcoides strains

The 16S rRNA gene provides insufficient information to infer the range of chloroorganic electron acceptors used by different Dehalococcoides organisms. To overcome this limitation and provide enhanced diagnostic tools for growth measurements, site assessment, and bioremediation monitoring, a quantitative real-time PCR (qPCR) approach targeting 16S rRNA genes and three Dehalococcoides reductive dehalogenase (RDase) genes with assigned function (i.e., tceA, bvcA, and vcrA) was designed and evaluated. qPCR standard curves generated for the RDase genes by use of genomic DNA from Dehalococcoides pure cultures correlated with standard curves obtained for both Bacteria- and Dehalococcoides-targeted 16S rRNA genes, suggesting that the RDase genes are useful targets for quantitative assessment of Dehalococcoides organisms. RDase gene probe/primer pairs were specific for the Dehalococcoides strains known to carry the diagnostic RDase gene sequences, and the qPCR method allowed the detection of as few as 1 to 20 and quantification of as few as 50 to 100 tceA, bvcA, or vcrA gene targets per PCR volume. The qPCR approach was applied to dechlorinating enrichment cultures, microcosms, and samples from a contaminated site. In characterized enrichment cultures where known Dehalococcoides strains were enumerated, the sum of the three RDase genes equaled the total Dehalococcoides cell numbers. In site samples and chloroethane-dechlorinating microcosms, the sum of the three RDase genes was much less than that predicted by Dehalococcoides-targeted qPCR, totaling 10 to 30% of the total Dehalococcoides cell numbers. Hence, a large number of Dehalococcoides spp. contain as-yet-unidentified RDase genes, indicating that our current understanding of the dechlorinating Dehalococcoides community is incomplete.     

Stepwise Kinetic Equilibrium Models of Quantitative Polymerase Chain Reaction

ABSTRACT: BACKGROUND: Numerous models for use in interpreting quantitative PCR (qPCR) data are present in recent literature. The most commonly used models assume the amplification in qPCR is exponential and fit an exponential model with a constant rate of increase to a select part of the curve. Kinetic theory may be used to model the annealing phase and does not assume constant efficiency of amplification. Mechanistic models describing the annealing phase with kinetic theory offer the most potential for accurate interpretation of qPCR data. Even so, they have not been thoroughly investigated and are rarely used for interpretation of qPCR data. New results for kinetic modeling of qPCR are presented. RESULTS: Two models are presented in which the efficiency of amplification is based on equilibrium solutions for the annealing phase of the qPCR process. Model 1 assumes annealing of complementary targets strands and annealing of target and primers are both reversible reactions and reach a dynamic equilibrium. Model 2 assumes all annealing reactions are nonreversible and equilibrium is static. Both models include the effect of primer concentration during the annealing phase. Analytic formulae are given for the equilibrium values of all single and double stranded molecules at the end of the annealing step. The equilibrium values are then used in a stepwise method to describe the whole qPCR process. Rate constants of kinetic models are the same for solutions that are identical except for possibly having different initial target concentrations. Analysis of qPCR curves from such solutions are thus analyzed by simultaneous non-linear curve fitting with the same rate constant values applying to all curves and each curve having a unique value for initial target concentration. The models were fit to two data sets for which the true initial target concentrations are known. Both models give better fit to observed qPCR data than other kinetic models present in the literature. They also give better estimates of initial target concentration. Model 1 was found to be slightly more robust than model 2 giving better estimates of initial target concentration when estimation of parameters was done for qPCR curves with very different initial target concentration. Both models may be used to estimate the initial absolute concentration of target sequence when a standard curve is not available. CONCLUSIONS: It is argued that the kinetic approach to modeling and interpreting quantitative PCR data has the potential to give more precise estimates of the true initial target concentrations than other methods currently used for analysis of qPCR data. The two models presented here give a unified model of the qPCR process in that they explain the shape of the qPCR curve for a wide variety of initial target concentrations.     

Real-time PCR detection of Holophagae (Acidobacteria) and Verrucomicrobia subdivision 1 groups in bulk and leek (Allium porrum) rhizosphere soils

In the light of the poor culturability of Acidobacteria and Verrucomicrobia species, group-specific real-time (qPCR) systems were developed based on the 16S rRNA gene sequences from culturable representatives of both groups. The number of DNA targets from three different groups, i.e. Holophagae (Acidobacteria group 8) and Luteolibacter/Prosthecobacter and unclassified Verrucomicrobiaceae subdivision 1, was determined in DNA extracts from different leek (Allium porrum) rhizosphere soil compartments and from bulk soil with the aim to determine the distribution of the three bacterial groups in the plant-soil ecosystem. The specificity of the designed primers was evaluated in three steps. First, in silico tests were performed which demonstrated that all designed primers 100% matched with database sequences of their respective groups, whereas lower matches with other non-target bacterial groups were found. Second, PCR amplification with the different primer sets was performed on genomic DNA extracts from target and from non-target bacteria. This test demonstrated specificity of the designed primers for the target groups, as single amplicons of expected sizes were found only for the target bacteria. Third, the qPCR systems were tested for specific amplifications from soil DNA extracts and 48 amplicons from each primer system were sequenced. All sequences were > 97% similar to database sequences of the respective target groups. Estimated cell numbers based on Holophagae-, Luteolibacter/Prosthecobacter- and unclassified Verrucomicrobiaceae subdivision 1-specific qPCRs from leek rhizosphere compartments and bulk soils demonstrated higher preference for one or both rhizosphere compartments above bulk soil for all three bacterial groups.     

Risk of Gastrointestinal Disease Associated with Exposure to Pathogens in the Water of the Lower Passaic River

During precipitation events, untreated human sewage is often intentionally discharged to surface water bodies via combined sewer overflow (CSO) systems in order to avoid overloading wastewater treatment plants. The purpose of this analysis was to evaluate the risk of pathogen-related disease associated with CSO discharges into the Lower Passaic River. Concentrations of fecal coliform, total coliform, fecal Streptococcus, and fecal Enterococcus bacteria were measured at six river locations on six different days in 2003 (n = 36). In addition, water samples (n = 2) were collected directly from and in the immediate vicinity of a discharging CSO in Newark, NJ. These samples were analyzed for fecal coliforms, total coliforms, fecal Streptococcus, fecal Enterococcus, Giardia lamblia, Cryptosporidium parvum, and several viruses. Risk estimates for gastrointestinal illness and Giardia infection resulting from indirect and direct ingestion of contaminated water were calculated for three potential exposure scenarios: visitor, recreator, and homeless person. Single-event risk was first evaluated for the three individual exposure scenarios; overall risk was then determined over a 1-year period. Monte Carlo techniques were used to characterize uncertainty. Nearly all of the pathogen concentrations measured in the Passaic River exceeded health-based water quality criteria and in some cases were similar to levels reported for raw sewage. The probability of contracting gastrointestinal illness due to fecal Streptococcus and Enterococcus from incidental ingestion of water over the course of a year ranged from 0.14 to nearly 0.70 for the visitor and recreator scenarios, respectively. For the homeless person exposure scenario, the risk for gastrointestinal illness reached 0.88 for fecal Streptococcus and Enterococcus, while the probability of Giardia infection was 1.0. This risk analysis suggests that, due to the levels of pathogens present in the Lower Passaic River, contact with the water poses, and will continue to pose, significant human health risks until CSO discharges are adequately controlled or abated.