Quantitative study of viable Vibrio parahaemolyticus cells in raw seafood using propidium monoazide in combination with quantitative PCR

In this study we developed a specific and sensitive quantitative PCR (qPCR) method combined with a propidium monoazide (PMA) sample treatment to quantify tdh-positive viable cells of V. parahaemolyticus in raw seafood (PMA-qPCR). The high selectivity of primers and probes were demonstrated by using purified DNA from 57 strains belonging to 18 species. Using these primers and probes for qPCR and in artificial contamination samples, a good correlation was obtained between Ct values and log CFU/reaction in the range of 12-1.2×10(6)CFU/reaction both from qPCR and PMA-qPCR with R(2) values of 0.9973 and 0.9919, respectively. The optimization of PMA concentration showed that 8 μg/mL was considered optimal to achieve a compromise between minimal impact on intact cells and maximal signal reduction in compromised cells. However, turbidity and cell concentration experiments showed that PMA treatment was not effective in samples where turbidities were ≥10 NTU and OD(600 nm) values were ≥0.8. PMA-qPCR was compared with culture isolation and traditional qPCR in environmental samples (including oyster, scallop, shrimp, and crab). The PMA-qPCR resulted in lower numbers of log CFUg(-1) than qPCR, with values having better agreement with numbers determined by culture isolation. In conclusion, this method is an effective tool for producing reliable quantitative data on viable V. parahaemolyticus in raw seafood.     

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.     

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.     

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.     

A Quantitative Real-Time PCR Method Using an X-Linked Gene for Sex Typing in Pigs

At present, a wide range of molecular sex-typing protocols in wild and domestic animals are available. In pigs, most of these methods are based on PCR amplification of X–Y homologous genes followed by gel electrophoresis which is time-consuming and in some cases expensive. In this paper, we describe, for the first time, a SYBR green-based quantitative real-time PCR (qPCR) assay using an X-linked gene, the glycoprotein M6B, for genetic sexing of pigs. Taking into account the differences in the glycoprotein M6B gene copy number between genders, we determine the correct sex of 54 pig samples from either diaphragm or hair follicle from different breeds using the 2^?ΔΔCT method for relative quantification. Our qPCR assay represents a quick, inexpensive, and reliable tool for sex determination in pigs. This new protocol could be easily adapted to other species in which the sex determination was required.     

A quantitative PCR method for measuring absolute telomere length

We describe a simple and reproducible method to measure absolute telomere length (aTL) using quantitative real-time polymerase chain reaction (qPCR). This method is based on the Cawthon method for relative measurement of telomere length (TL) but modified by introducing an oligomer standard to measure aTL. The method describes the oligomer standards, the generation of the standard curve and the calculations required to calculate aTL from the qPCR data. The necessary controls and performance characteristics of the assay are described in detail and compared relative to other methods for measuring TL. Typical results for this assay for a variety of human tissue samples are provided as well as a troubleshooting schedule. This method allows high throughput measurement of aTL using small amounts of DNA making it amenable for molecular epidemiological studies. Compared to the traditional relative TL qPCR assays, the aTL method described in this protocol enables a more direct comparison of results between experiments within and between laboratories.     

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.     

Inactivation of enteric adenovirus and feline calicivirus by chlorine dioxide

Chlorine dioxide (ClO2) inactivation experiments were conducted with adenovirus type 40 (AD40) and feline calicivirus (FCV). Experiments were carried out in buffered, disinfectant demand-free water under high- and low-pH and -temperature conditions. Ct values (the concentration of ClO2 multiplied by contact time with the virus) were calculated directly from bench-scale experiments and from application of the efficiency factor Hom (EFH) model. AD40 Ct ranges for 4-log inactivation (Ct99.99%) at 5 degrees C were >0.77 to <1.53 mg/liter x min and >0.80 to <1.59 mg/liter x min for pH 6 and 8, respectively. For 15 degrees C AD40 experiments, >0.49 to <0.74 mg/liter x min and <0.12 mg/liter x min Ct99.99% ranges were observed for pH 6 and 8, respectively. FCV Ct99.99% ranges for 5 degrees C experiments were >20.20 to <30.30 mg/liter x min and >0.68 mg/liter x min for pH 6 and 8, respectively. For 15 degrees C FCV experiments, Ct99.99% ranges were >4.20 to <6.72 and <0.18 mg/liter x min for pH 6 and 8, respectively. Viral inactivation was higher at pH 8 than at pH 6 and at 15 degrees C than at 5 degrees C. Comparison of Ct values and inactivation curves demonstrated that the EFH model described bench-scale experiment data very well. Observed bench-scale Ct99.99% ranges and EFH model Ct99.99% values demonstrated that FCV is more resistant to ClO2 than AD40 for the conditions studied. U.S. Environmental Protection Agency guidance manual Ct99.99% values are higher than Ct99.99% values calculated from bench-scale experiments and from EFH model application.     

DNA甲基化分析中重亚硫酸盐处理DNA转化效率的评估方法

为建立一种评估重亚硫酸盐处理DNA样本后胞嘧啶转化效率的有效方法,以两组不同的TaqMan qPCR检测梯度稀释的重亚硫酸盐处理和未处理的DNA标准品,建立转化与未转化的DNA Ct值以及对应的DNA拷贝数的标准曲线。使用相同的探针定量检测重亚硫酸盐处理后的DNA样本评估转化效率。结果显示该方法应用两组探针,根据相应的标准曲线,精确评估样本经重亚硫酸盐处理的转化效率。使用已知转化和未转化拷贝数的混合DNA作为模板,证实了该方法的可靠性。同时也对不同重亚硫酸盐试剂盒处理DNA的转化效率进行了评估,结果显示,该方法能够有效地评估DNA样品重亚硫酸盐的转化效率,为DNA甲基化准确分析提供了可靠快捷的方法。     

Fibrinogen and the early stages of polymerization to fibrin as studied by dynamic laser light scattering

Human fibrinogen and the polymerization of fibrin after activation by the enzymes, thrombin and Batroxobin, were studied by means of dynamic laser light scattering (DLS). The apparent diffusion constant, D, for fibrinogen was measured and has a value of (1.80 +/- 0.42) X 10(-7) cm2 X s-1. D was found to contain contributions from the translational diffusion constant (Dt) as well as from the rotational diffusion constant (Dr). A comparison between experimental and calculated values of Dr and Dt suggests that fibrinogen in the absence of added Ca2+ expresses a certain degree of flexibility, while it is straightened in the presence of added Ca2+. The time dependence of D showed periodic oscillations, while the average D values decreased with time. Thrombin and Batroxobin caused similar behaviour of D. The period length was related to the enzyme concentration, clotting time (Ct) and the rate of release of fibrinopeptide A (FPA). No periodic oscillations were observed in experiments where the enzyme was replaced by saline, or in experiments using a dysfunctional fibrinogen (fibrinogen Aarhus) which displayed slow rates of FPA-release and polymerization. We propose that the periodic oscillations in a system far from equilibrium may be explained by conformational changes occurring in the fibrinogen molecule during enzyme activation and polymerization.     

Use of real-time PCR for determining copy number and zygosity in transgenic plants

This review examines how real-time PCR can be used to determine copy number and zygosity in transgenic plants. Distinguishing between plants that harbor one and two copies of a transgene or are hemizygous and homozygous requires the ability to routinely distinguish twofold differences, a detection difference which approaches the resolution of PCR-based quantification methods. After explaining the basic principles, especially the threshold cycle (Ct value) as the basic measuring unit in real-time PCR, we introduce three quantitation methods currently in use. While the absolute and relative standard curve approaches are qualitative methods that distinguish high-copy from low-copy transformants, the comparative ( ) method with double-dye oligonucleotides (TaqMan probes) is able to detect twofold differences. In order to obtain reliable results, Ct values for an amplicon should be below 25 and the standard deviation below 0.3. Although real-time PCR can deliver exact copy number determinations, the procedure is not fail-safe. Therefore, real-time PCR should to be viewed as complementary to—rather than as a replacement of—other methods such as Southern analysis, but it is particularly useful as a preliminary screening tool for estimating copy numbers of a large number of transformants.     

Chlorine inactivation of adenovirus type 40 and feline calicivirus

Ct values, the concentration of free chlorine multiplied by time of contact with virus, were determined for free-chlorine inactivation experiments carried out with chloroform-extracted (dispersed) and non-chloroform-extracted (aggregated) feline calicivirus (FCV), adenovirus type 40 (AD40), and polio virus type 1 (PV-1). Experiments were carried out with high and low pH and temperature conditions. Ct values were calculated directly from bench-scale free-chlorine inactivation experiments and from application of the efficiency factor Hom model. For each experimental condition, Ct values were higher at pH 8 than at pH 6, higher at 5 degrees C than at 15 degrees C, and higher for dispersed AD40 (dAD40) than for dispersed FCV (dFCV). dFCV and dAD40 were more sensitive to free chlorine than dispersed PV-1 (dPV-1). Cts for 2 log inactivation of aggregated FCV (aFCV) and aggregated PV-1 (aPV-1) were 31.0 and 2.8 orders of magnitude higher than those calculated from experiments carried out with dispersed virus. Cts for 2 log inactivation of dFCV and dAD40 in treated groundwater at 15 degrees C were 1.2 and 13.7 times greater than in buffered-demand-free (BDF) water experiments at 5 degrees C. Ct values listed in the U.S. Environmental Protection Agency (EPA) Guidance Manual were close to, or lower than, Ct values generated for experiments conducted with dispersed and aggregated viruses suspended in BDF water and for dispersed viruses suspended in treated groundwater. Since the state of viruses in water is most likely to be aggregated and associated with organic or inorganic matter, reevaluation of the EPA Guidance Manual Ct values is necessary, since they would not be useful for ensuring inactivation of viruses in these states. Under the tested conditions, dAD40, dFCV, aFCV, dPV-1, and aPV-1 particles would be inactivated by commonly used free chlorine concentrations (1 mg/liter) and contact times (60 to 237 min) applied for drinking water treatment in the United States.     

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.     

Determination of the molecular mass of bacterial genomic DNA and plasmid copy number by high-pressure liquid chromatography

Relatively rapid methods for the determination of relative genome molecular mass (Mr) and the estimation of plasmid copy number have been developed. These methods are based on the ability of the Bio-Rad high-pressure liquid chromatography hydroxylapatite column to separate and quantify single-stranded DNA, double-stranded DNA, and plasmid DNA. Genome Mr values were calculated from reassociation kinetics of single-stranded DNA as measured with the hydroxylapatite column. Bacteriophage T4 DNA was used to establish a C0t (moles of nucleotides times seconds per liter), or standard reassociation value. From this C0t value, C0t values for Escherichia coli B, Beggiatoa alba B18LD, and Streptomyces coelicolor were determined by comparative calculations. From those calculated C0t values, the Mr values of 1.96 X 10(9) for E. coli, 2.02 X 10(9) for B. alba, and 3.28 X 10(9) for S. coelicolor were estimated. Plasmid concentration was determined from cleared lysates by comparing the integrated area under the phosphate buffer-eluted plasmid peak to values obtained with known amounts of plasmid. The plasmid copy number was estimated by multiplying the ratio between the amounts of plasmid and chromosomal DNA by the ratio between the Mr values of the chromosome and the plasmid. A copy number of 29 was obtained from a culture of E. coli HB101 harboring pBR322 grown to a culture density of 1.6 X 10(9) CFU . ml-1.     

Interaction of water vapour with twenty different poly-L-amino acids and their excess hydration in presence of sodium chloride

Using the isopiestic vapour pressure technique, the magnitudes of excess binding of water and NaCl per mole of twenty different poly-L-amino acid residues, respectively in the presence of different bulk molefractions (X2) of NaCl have been evaluated from the mathematical expressions for the Gibbs surface excesses. At certain high ranges of NaCl concentration, the plot of -Gamma1 (2) versus X1/X2 becomes linear, so that moles of water and NaCl, respectively bound per mole of amino acid residue can be evaluated. -Gamma(2)1 is the excess moles of H20 per mole of amino acid residue and X1 and X2 stand for mole fractions of the water and NaCl, respectively in the sample system. Also, using the integrated form of the Gibbs absorption equation, the values of standard free energy change (deltaG(0)) for the excess adsorption of NaCl per kg of poly-L-amino acids have been evaluated. These values are all positive as a result of positive excess hydration of polyamino acids. The standard free energy of excess hydration deltaG(0)hy (equal to -deltaG(0)) is negative due to spontaneous excess hydration of polyamino acid in the presence of a salt.     

Determination of the molecular mass of bacterial genomic DNA and plasmid copy number by high-pressure liquid chromatography.

Relatively rapid methods for the determination of relative genome molecular mass (Mr) and the estimation of plasmid copy number have been developed. These methods are based on the ability of the Bio-Rad high-pressure liquid chromatography hydroxylapatite column to separate and quantify single-stranded DNA, double-stranded DNA, and plasmid DNA. Genome Mr values were calculated from reassociation kinetics of single-stranded DNA as measured with the hydroxylapatite column. Bacteriophage T4 DNA was used to establish a C0t (moles of nucleotides times seconds per liter), or standard reassociation value. From this C0t value, C0t values for Escherichia coli B, Beggiatoa alba B18LD, and Streptomyces coelicolor were determined by comparative calculations. From those calculated C0t values, the Mr values of 1.96 X 10(9) for E. coli, 2.02 X 10(9) for B. alba, and 3.28 X 10(9) for S. coelicolor were estimated. Plasmid concentration was determined from cleared lysates by comparing the integrated area under the phosphate buffer-eluted plasmid peak to values obtained with known amounts of plasmid. The plasmid copy number was estimated by multiplying the ratio between the amounts of plasmid and chromosomal DNA by the ratio between the Mr values of the chromosome and the plasmid. A copy number of 29 was obtained from a culture of E. coli HB101 harboring pBR322 grown to a culture density of 1.6 X 10(9) CFU . ml-1.     

Accurate Determination of Zygosity in Transgenic Rice by Real-time PCR Does Not Require Standard Curves or Efficiency Correction

A number of quantitative, real-time PCR methods have been developed for determining transgene copy numbers in plants. Here, we demonstrate that the Roche LightCycler^TM system can be used to determine the zygosity of transgenic lines without the use of standard curves or efficiency correction calculations. We have developed a duplex PCR assay which permits the determination of zygosity, relative to a calibrator sample, in transgenic rice lines containing the gene for a viral glycoprotein. Our data demonstrate that unambiguous 2-fold discrimination of copy number can be attained by calculating relative copy number using the threshold crossing point (Ct) calculated by the LightCycler^TM software combined with delta delta Ct calculations, provided that the appropriate calibrator sample is included in each run. The method presented here is rapid, sensitive, robust and easy to optimise.     

Standards and predicted values of anaerobic threshold

Mean values for body size, body composition and endurance indices have been obtained from a homogeneous group of 125 physically active men to find predicted values of AT (age 23.4 +/- 4.3 years; height 175.9 +/- 6.5 cm; weight 72.2 +/- 8.9 kg; body fat 17.9 +/- 4.7% body weight, muscularity index 19.0 +/- 1.5 kg fat-free mass/cm2 X 10(-4) height; forced vital lung capacity 5667 +/- 815 cm3; VO2max 48.5 +/- 6.0 cm3 X kg-1 X min-1; anaerobic threshold 61.0 +/- 7.8% VO2max). Endurance performance and fitness indices were a little higher than average, but about 10% lower than in endurance-trained athletes. The authors suggest that standards of anaerobic threshold (AT) for ergonomics and endurance training should be about 55-65% VO2max, but not lower than 1800 cm3 O2 X min-1. The coefficients of correlation of AT relating to VO2max, PFO2 and submaximal load were significant at the 0.01 level. Using regression analysis, predicted values of AT were developed. A predicted value of AT can be obtained from the regression line of AT on Lsubmax used as a nomogram, during a simple PWC170 exercise test without blood or gas analysis.