Evaluation of uncertainty in quantitative real-time PCR

Quantitative real-time PCR is one of the newer methods for measurement of the amount of nucleic material in biological systems. However, reliable measurement requires an appropriate estimation of uncertainty and this paper has developed the uncertainty budget associated with this procedure using as an example, data from a quantitative real-time PCR method for the enumeration of Campylobacter jejuni. This uncertainty is relatively large and for instance, a measured result of 151 units of DNA would have a 95% confidence interval of +/-84 units of DNA with the main sources of uncertainty being the measurement of the threshold cycle (Ct) value, the predicted DNA content of the unknown sample from the calibration line and the molar absorbance value for DNA.     

Adaptation of a real-time PCR method for the detection and quantification of pathogenic leptospires in environmental water

Leptospirosis is a major zoonotic disease that affects humans and animals in all continents, in both rural and urban areas. In Europe, metropolitan France is the most affected country, with about 300 human cases declared per year. In France, although leptospirosis is now mostly considered as a recreational disease related to freshwater areas, isolation of pathogenic leptospires from environmental water samples still remains difficult. It thus seemed important to set up an efficient method to detect and quantify these bacteria in this environment. We determined a DNA extraction method suitable for freshwater samples and adapted a real-time quantitative PCR based on the detection of the LipL32 gene using the SYBR green chemistry. The method developed is specific for pathogenic Leptospira. It permits the detection of all the pathogenic strains tested and none of the saprophytic strains. Quantification is possible between 10 and 10(7) bacteria/mL, and therefore, the method represents a tool that could be integrated into future public health surveillance programs for recreational freshwater areas.     

Application of quantitative real-time PCR for enumeration of total bacterial,archaeal, and yeast populations in kimchi

Kimchi is a Korean traditional fermented food made of brined vegetables, with a variety of spices. Various microorganisms are associated with the kimchi fermentation process. This study was undertaken in order to apply quantitative real-time PCR targeting the 16S and 26S rRNA genes for the investigation of dynamics of bacterial, archaeal, and yeast communities during fermentation of various types of kimchi. Although the total bacterial and archaeal rRNA gene copy numbers increased during kimchi fermentation, the number of yeasts was not significantly altered. In 1 ng of bulk DNA, the mean number of rRNA gene copies for all strains of bacteria was 5.45×10⁶ which was 360 and 50 times greater than those for archaea and yeast, respectively. The total gene copy number for each group of microorganisms differed among the different types of kimchi, although the relative ratios among them were similar. The common dominance of bacteria in the whole microbial communities of various types of kimchi suggests that bacteria play a principal role in the kimchi fermentation process.     

Development and evaluation of a Taqman duplex real-time PCR quantification method for reliable enumeration of Legionella pneumophila in water samples

This study describes the development and evaluation of a specific Legionella pneumophila Taqman duplex real-time PCR (qPCR) for fast and reliable quantification of this human pathogen in suspected man-made water systems. The qPCR assay was 100% specific for all L. pneumophila serogroups 1-15 with a sensitivity of 60 genome units/l and an amplification efficiency of 98%. Amplification inhibitors were detected via an exogenous internal positive control, which was amplified simultaneously with L. pneumophila DNA using its own primer and probe set. Mean recovery rates of the qPCR assay for tap water and cooling circuit water, spiked with a known number L. pneumophila bacteria, were 93.0% and 56.3%, respectively. Additionally, by using the Ultraclean Soil DNA isolation kit, we were able to remove amplification inhibitors ubiquitously present in cooling water. The practical value of our qPCR assay was evaluated through analysis of 30 water samples from showers, taps, eyewash stations, fire sprinklers and recirculation loops with qPCR and traditional culture. In conclusion, the described L. pneumophila Taqman duplex real-time assay proved to be specific, sensitive and reproducible. This makes it a promising method complementing the current time-consuming culture standard method.     

Evaluation of reference genes for real-time quantitative PCR studies in

ABSTRACT: BACKGROUND: The selection of stable and suitable reference genes for real-time quantitative PCR (RT-qPCR) is a crucial prerequisite for reliable gene expression analysis under different experimental conditions. The present study aimed to identify reference genes as internal controls for gene expression studies by RT-qPCR in azole-stimulated Candida glabrata. RESULTS: The expression stability of 16 reference genes under fluconazole stress was evaluated using fold change and standard deviation computations with the hkgFinder tool. Our data revealed that the mRNA expression levels of three ribosomal RNAs (RDN5.8, RDN18, and RDN25) remained stable in response to fluconazole, while PGK1, UBC7, and UBC13 mRNAs showed only approximately 2.9-, 3.0-, and 2.5-fold induction by azole, respectively. By contrast, mRNA levels of the other 10 reference genes (ACT1, EF1a, GAPDH, PPIA, RPL2A, RPL10, RPL13A, SDHA, TUB1, and UBC4) were dramatically increased in C. glabrata following antifungal treatment, exhibiting changes ranging from 4.5- to 32.7-fold. We also assessed the expression stability of these reference genes using the 2-[increment][increment]CT method and three other software packages. The stability rankings of the reference genes by geNorm and the 2-[increment][increment]CT method were identical to those by hkgFinder, whereas the stability rankings by BestKeeper and NormFinder were notably different. We then validated the suitability of six candidate reference genes (ACT1, PGK1, RDN5.8, RDN18, UBC7, and UBC13) as internal controls for ten target genes in this system using the comparative CT method. Our validation experiments passed for all six reference genes analyzed except RDN18, where the amplification efficiency of RDN18 was different from that of the ten target genes. Finally, we demonstrated that the relative quantification of target gene expression varied according to the endogenous control used, highlighting the importance of the choice of internal controls in such experiments. CONCLUSIONS: We recommend the use of RDN5.8, UBC13, and PGK1 alone or the combination of RDN5.8 plus UBC13 or PGK1 as reference genes for RT-qPCR analysis of gene expression in C. glabrata following azole treatment. In contrast, we show that ACT1 and other commonly used reference genes (GAPDH, PPIA, RPL13A, TUB1, etc.) were not validated as good internal controls in the current model.     

A quantitative real-time PCR method for absolute telomere length

Telomere shortening is an important risk factor for cancer and accelerated aging. Here we describe the development of a simple and reproducible method to measure absolute telomere length. Based on Cawthon's quantitative real-time PCR (qRT-PCR) assay, our method uses an oligomer standard that can be used to generate absolute telomere length values rather than relative quantification. We demonstrate a strong correlation between this improved method and the "gold standard" of telomere length measurement-terminal restriction fragment analysis (TRF) by Southern hybridization. The capability to generate absolute telomere length values should allow a more direct comparison of results between experiments within and between laboratories.     

Evaluation of a rapid, defined substrate technology method for enumeration of total coliforms and Escherichia coli in chlorinated drinking water

A rapid, defined substrate technology method, commercially available as Colilert, simultaneously enumerates total coliforms and Escherichia coli in drinking water samples in 24 h without the need for confirmatory tests. The ability of this method to enumerate both total coliforms and E. coli in simulated chlorine-treated drinking water samples was compared with the standard UK method (minerals-modified glutamate most probable number) which requires up to 96 h to complete including confirmation. Statistical analysis by a nonparametric matched-pair test showed the Colilert method to be less efficient at detecting down to one E. coli in these samples compared to the standard UK method. No statistically significant difference between the two methods of enumeration for total coliforms was detected.     

Evaluation of methods for oligonucleotide array data via quantitative real-time PCR

Background  

There are currently many different methods for processing and summarizing probe-level data from Affymetrix oligonucleotide arrays. It is of great interest to validate these methods and identify those that are most effective. There is no single best way to do this validation, and a variety of approaches is needed. Moreover, gene expression data are collected to answer a variety of scientific questions, and the same method may not be best for all questions. Only a handful of validation studies have been done so far, most of which rely on spike-in datasets and focus on the question of detecting differential expression. Here we seek methods that excel at estimating relative expression. We evaluate methods by identifying those that give the strongest linear association between expression measurements by array and the "gold-standard" assay.     

Development of a new real-time TaqMan PCR assay for quantitative analyses of Candida albicans resistance genes expression

Candida albicans is an important opportunistic pathogen that can cause serious fungal diseases in immunocompromised patients including cancer patients, transplant patients, and patients receiving immunosuppressive therapy in general, those with human immunodeficiency virus infections and undergoing major surgery. Its emergence spectrum varies from mucosal to systemic infections and the first line treatment is still based on fluconazole, a triazole derivate with a potent antifungal activity against most of C. albicans strains. Nevertheless the emergence of fluconazole-resistant C. albicans strains can lead to treatment failures and thus become a clinical problem in the management of such infections. For that reason we consider it important to study mechanisms inducing azole resistance and the possibilities to influence this process. In this work we give a short report on a real-time PCR (TaqMan) assay, which can be used for quantitative analyses of gene expression levels of MDR1, CDR1 and ERG11, genes supposed to contribute to development of the resistance mechanisms. We show some results achieved with that assay in fluconazole susceptible and resistant strains that confirm results seen earlier in experiments using Northern blot hybridisation and prove that the comparative DeltaCt method is valid for our system.     

Rapid identification and enumeration of Saccharomyces cerevisiae cells in wine by real-time PCR

Despite the beneficial role of Saccharomyces cerevisiae in the food industry for food and beverage production, it is able to cause spoilage in wines. We have developed a real-time PCR method to directly detect and quantify this yeast species in wine samples to provide winemakers with a rapid and sensitive method to detect and prevent wine spoilage. Specific primers were designed for S. cerevisiae using the sequence information obtained from a cloned random amplified polymorphic DNA band that differentiated S. cerevisiae from its sibling species Saccharomyces bayanus, Saccharomyces pastorianus, and Saccharomyces paradoxus. The specificity of the primers was demonstrated for typical wine spoilage yeast species. The method was useful for estimating the level of S. cerevisiae directly in sweet wines and red wines without preenrichment when yeast is present in concentrations as low as 3.8 and 5 CFU per ml. This detection limit is in the same order as that obtained from glucose-peptone-yeast growth medium (GPY). Moreover, it was possible to quantify S. cerevisiae in artificially contaminated samples accurately. Limits for accurate quantification in wine were established, from 3.8 x 10(5) to 3.8 CFU/ml in sweet wine and from 5 x 10(6) to 50 CFU/ml in red wine.     

Evaluation of progesterone receptor expression in eosinophils using real-time quantitative PCR

Progesterone has been shown in many instances to have immune-suppressant activities. Most of these activities have been investigated in the light of general immune suppression or with a focus on lymphocytes. However, many clinical and in vitro studies have shown that progesterone also has a suppressive effect on eosinophilia. This effect so far has not been thoroughly investigated. The purpose of this study was to evaluate whether the effect is mediated via the classical progesterone receptor (PR). We developed a new real-time quantitative PCR (RQ-PCR) for the analysis and quantification of expression of the classical PR. The test was first validated both on breast cancer cell lines and on breast cancer biopsies. Subsequently, when using eosinophils isolated from peripheral blood of healthy volunteers, we could not find evidence for the expression of PR. These data suggest that the effects of progesterone on eosinophils are not mediated by the classical PR.     

A one-step method for quantitative determination of uracil in DNA by real-time PCR

Uracil may occur in DNA due to either cytosine deamination or thymine replacing incorporation. Its quantitative characterization is important in assessing DNA damages in cells with perturbed thymidylate metabolism or within different DNA segments involved in immunoglobulin gene diversification. The archaeal DNA polymerase from Pyrococcus furiosus binds strongly to the deaminated base uracil and stalls on uracil-containing templates. Here, we present a straightforward method for quantitative assessment of uracil in DNA within specific genomic segments. We use wild-type P. furiosus polymerase in parallel with its point mutant version which lacks the uracil-binding specificity on synthetic and genomic DNA samples to quantify the uracil content in a single-step real-time PCR assay. Quantification of the PCR results is based on an approach analogous to template copy number determination in comparing different samples. Data obtained on synthetic uracil-containing templates are verified by direct isotopic measurements. The method is also tested on physiological DNA samples from Escherichia coli and mouse cell lines with perturbed thymidylate biosynthesis. The present PCR-based method is easy to use and measures the uracil content within a genomic segment defined by the primers. Using distinct sets of primers, the method allows the analysis of heterogeneity of uracil distribution within the genome.     

A quantitative real-time PCR method for in planta monitoring of Phytophthora infestans growth

Aim: To develop a real‐time PCR‐based strategy for the detection of Paenibacillus larvae vegetative cells and spores to improve the diagnosis and the screening of American foulbrood (AFB), the most harmful pathology of honeybee brood. Methods and Results: A real‐time PCR that allowed selective identification and quantification of P. larvae 16S rRNA sequence was developed. Using standard samples quantified by flow cytometry, detection limits of 37·5 vegetative cells ml^?1 and 10 spores ml^?1 were determined. Compared to spread plate method, this real‐time PCR‐based strategy allowed, in only 2 h, the detection of P. larvae in contaminated honeys. No false‐positive results were obtained. Moreover, its detection limit was 100 times lower than that of the culture method (2 vs 200 spores g^?1 of honey). Conclusion: A rapid, selective, with low detection limit, sensitive and specific method to detect and quantify vegetative cells and spores of P. larvae is now available. Significance and Impact of Study: In addition to honey samples, this real‐time PCR‐based strategy may be also applied to confirm AFB diagnosis in honeybee brood and to screen other apiary supplies and products (bees, pollen, wax), thus broadening the control of AFB spreading.     

A new method for robust quantitative and qualitative analysis of real-time PCR

An automated data analysis method for real-time PCR needs to exhibit robustness to the factors that routinely impact the measurement and analysis of real-time PCR data. Robust analysis is paramount to providing the same interpretation for results regardless of the skill of the operator performing or reviewing the work. We present a new method for analysis of real-time PCR data, the maxRatio method, which identifies a consistent point within or very near the exponential region of the PCR signal without requiring user intervention. Compared to other analytical techniques that generate only a cycle number, maxRatio generates several measurements of amplification including cycle numbers and relative measures of amplification efficiency and curve shape. By using these values, the maxRatio method can make highly reliable reactive/nonreactive determination along with quantitative evaluation. Application of the maxRatio method to the analysis of quantitative and qualitative real-time PCR assays is shown along with examples of method robustness to, and detection of, amplification response anomalies.     

Diagnostics method for the rapid quantitative detection and identification of low-level contamination of high-purity water with pathogenic bacteria

High-purity water (HPW) can be contaminated with pathogenic microorganisms, which may result in human infection. Current culture-based techniques for the detection of microorganisms from HPW can be slow and laborious. The aim of this study was to develop a rapid method for the quantitative detection and identification of pathogenic bacteria causing low-level contamination of HPW. A novel internally controlled multiplex real-time PCR diagnostics assay was designed and optimized to specifically detect and identify Pseudomonas aeruginosa and the Burkholderia genus. Sterile HPW, spiked with a bacterial load ranging from 10 to 10³ cfu/100 ml, was filtered and the bacterial cells were removed from the filters by sonication. Total genomic DNA was then purified from these bacteria and subjected to testing with the developed novel multiplex real-time PCR diagnostics assay. The specific P. aeruginosa and Burkholderia genus assays have an analytical sensitivity of 3.5 genome equivalents (GE) and 3.7 GE, respectively. This analysis demonstrated that it was possible to detect a spiked bacterial load of 1.06 × 10² cfu/100 ml for P. aeruginosa and 2.66 × 10² cfu/100 ml for B. cepacia from a 200-ml filtered HPW sample. The rapid diagnostics method described can reliably detect, identify, and quantify low-level contamination of HPW with P. aeruginosa and the Burkholderia genus in <4 h. We propose that this rapid diagnostics method could be applied to the pharmaceutical and clinical sectors to assure the safety and quality of HPW, medical devices, and patient-care equipment.     

Development of a SYBR green real-time PCR method for rapid detection of sheep pox virus

Background

Virus subtype H13N2, A/mallard/Kr/SH38-45/2010 (H13N2), was first isolated from a mallard fecal sample in South Korea.

Results

Phylogenetic analysis of all eight viral genes revealed that this virus emerged by genetic mixing between Eurasian and North American gene pools, and possibly between wild ducks and gulls. The H13 and N2 surface genes clustered together in a group with Eurasian isolates from gulls and wild birds, respectively. The PB2, PA, NP, M and NS segments belonged to the Eurasian lineage, whereas the PB1 gene clustered in the North American lineage. Furthermore, they showed a bird-dependent pattern in phylogenetic analysis: the M gene was similar to subtype H13 viruses within gulls, whereas other segments were similar to avian influenza viruses of other subtypes from wild ducks.

Conclusions

The data suggests that the novel reassortant H13N2 virus isolated in South Korea might have emerged by genetic reassortment between intercontinental and interspecies transmission in wild birds.     

临床皮炎外瓶霉荧光定量PCR检测方法的建立

目的 建立基于TaqMan探针技术的皮炎外瓶霉荧光定量PCR检测方法.方法 通过对皮炎外瓶霉ITS区域基因组序列（GenBank：JN675373.1）进行分析,设计合成特异性引物和荧光标记探针,优化荧光定量PCR反应条件.以临床标本中分离的皮炎外瓶霉为阳性菌株,及其他种类真菌和细菌作为阴性对照菌株,从特异性、敏感性及重复性方面对该方法检测效果进行评价.结果 该研究设计的引物和探针能扩增皮炎外瓶霉特异性序列.临床分离得到的皮炎外瓶霉在反应中有明显扩增曲线,而甄氏外瓶霉、棘状外瓶霉、烟曲霉、白色念珠菌、新生隐球菌、马内菲青霉等20株菌在CT值≤38范围内均未有扩增;利用基因重组构建的标准品完成了标准曲线的绘制,在1.0×103～1.0×107拷贝数（Cp）内具有良好的线性关系（R2=1.000）,最低可检出量为10 Cp/μL.结论 成功建立了荧光定量PCR检测皮炎外瓶霉方法,该法特异度强、敏感度高、重复性好,将有助于临床皮炎外瓶霉感染的早期诊断和针对性治疗.     

Application of real-time PCR for quantitative detection of Campylobacter jejuni in poultry, milk and environmental water

Campylobacter jejuni is a leading human food-borne pathogen. The rapid and sensitive detection of C. jejuni is necessary for the maintenance of a safe food/water supply. In this article, we present a real-time polymerase chain reaction (PCR) assay for quantitative detection of C. jejuni in naturally contaminated poultry, milk and environmental samples without an enrichment step. The whole assay can be completed in 60 min with a detection limit of approximately 1 CFU. The standard curve correlation coefficient for the threshold cycle versus the copy number of initial C. jejuni cells was 0.988. To test the PCR system, a set of 300 frozen chicken meat samples, 300 milk samples and 300 water samples were screened for the presence of C. jejuni. 30.6% (92/300) of chicken meat samples, 27.3% (82/300) of milk samples, and 13.6% (41/300) of water samples tested positive for C. jejuni. This result indicated that the real-time PCR assay provides a specific, sensitive and rapid method for quantitative detection of C. jejuni. Moreover, it is concluded that retail chicken meat, raw milk and environmental water are commonly contaminated with C. jejuni and could serve as a potential risk for consumers in eastern China, especially if proper hygienic and cooking conditions are not maintained.     

Assessment of EvaGreen-based quantitative real-time PCR assay for enumeration of the microalgae Heterosigma and Chattonella (Raphidophyceae)

Heterosigma akashiwo and Chattonella species (Raphidophyceae) are difficult to detect and quantify in environmental samples because of their pleomorphic and fragile cell nature. In this study, we developed a quantitative real-time polymerase chain reaction (qRT-PCR) assay for the enumeration of these algal taxa using a new DNA-binding dye, EvaGreen. Species-specific qRT PCR primers to H. akashiwo, Chattonella antiqua, Chattonella marina, Chattonella ovata, and Chattonella subsalsa were designed to target the ITS2 rRNA gene intergenic region. Primer specificities were tested via BLAST searches. In addition, specificity was verified using empirical tests, including competitive PCR. The qRT PCR assay analyzing C _t value and the log of cell number showed a significant linear relationship (r ²?≥?0.997). When light microscopy was used to monitor the population dynamics of targeted Raphidophyceae from Lake Shihwa, H. akashiwo was detected in ten samples and no Chattonella spp. were detected (70 samples collected from May, 2007 to January, 2008). In contrast, when the qRT-PCR assay was used, H. akashiwo was detected in 41 samples. C. antiqua, C. marina, and C. ovata were detected in eight samples. Most of the samples analyzed using qRT-PCR assays showed higher algal numbers than did those assayed via microscopy, suggesting that the enumeration of Raphidophyceae via classic microscopic methods most likely underestimates true algal concentration.