Browse
Submit Data
Databases
API
Help

Dataset Information

8 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Quantitation of viral DNA by real-time PCR applying duplex amplification, internal standardization, and two-color fluorescence detection.

ABSTRACT: A real-time PCR method was developed to quantitate viral DNA that includes duplex amplification, internal standardization, and two-color fluorescence detection without the need to generate an external standardization curve. Applied to human parvovirus B19 DNA, the linear range was from 10(2) to at least 5 x 10(6) copies per ml of sample. The coefficient of variation was 0.29 using a run control of 2,876 copies per ml. The method reduces the risk of false-negative results, yields high precision, and is applicable for other DNA targets.

SUBMITTER: Gruber F

PROVIDER: S-EPMC92947 | biostudies-literature | 2001 Jun

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Quantitation of viral DNA by real-time PCR applying duplex amplification, internal standardization, and two-color fluorescence detection.

Gruber F F Falkner F G FG Dorner F F Hämmerle T T

Applied and environmental microbiology 20010601 6

A real-time PCR method was developed to quantitate viral DNA that includes duplex amplification, internal standardization, and two-color fluorescence detection without the need to generate an external standardization curve. Applied to human parvovirus B19 DNA, the linear range was from 10(2) to at least 5 x 10(6) copies per ml of sample. The coefficient of variation was 0.29 using a run control of 2,876 copies per ml. The method reduces the risk of false-negative results, yields high precision, ...[more]

PMID: 11375203

Similar Datasets

Detection, differentiation, and quantitation of pathogenic leishmania organisms by a fluorescence resonance energy transfer-based real-time PCR assay.

Project description:Real-time technology eliminates many of the pitfalls of diagnostic PCR, but this method has not been applied to differentiation of Leishmania organisms so far. We have developed a real-time PCR that simultaneously detects, quantitates, and categorizes Leishmania organisms into three relevant groups causing distinct clinical pictures. The analytical sensitivity (detection rate of >or=95% at 94.1 parasites/ml of blood) was within a range that has been determined previously to facilitate the confirmation of visceral leishmaniasis from peripheral blood. Parasites were successfully detected in 12 different clinical samples (blood, bone marrow, skin, and liver). The Leishmania donovani complex, the Leishmania brasiliensis complex, and species other than these could be clearly discriminated by means of distinct melting temperatures obtained with fluorescence resonance energy transfer probes (melting points, 72.7, 67.1, and 65.0 degrees C, respectively). All three groups could be quantified within equal ranges. As in other real-time PCRs, the variability in the quantification of DNA was small (coefficient of variation [CV], <2%). However, human samples containing low levels of parasites (100 parasites per ml of blood) showed higher variation (CV, 60.89%). Therefore, despite its superior analytical performance, care must be taken when real-time PCR is utilized for therapy monitoring.

| S-EPMC153923 | biostudies-literature

A novel real-time PCR for Listeria monocytogenes that monitors analytical performance via an internal amplification control.

Project description:We describe a novel quantitative real-time (Q)-PCR assay for Listeria monocytogenes based on the coamplification of a target hly gene fragment and an internal amplification control (IAC). The IAC is a chimeric double-stranded DNA containing a fragment of the rapeseed BnACCg8 gene flanked by the hly-specific target sequences. This IAC is detected using a second TaqMan probe labeled with a different fluorophore, enabling the simultaneous monitoring of the hly and IAC signals. The hly-IAC assay had a specificity and sensitivity of 100%, as assessed using 49 L. monocytogenes isolates of different serotypes and 96 strains of nontarget bacteria, including 51 Listeria isolates. The detection and quantification limits were 8 and 30 genome equivalents, and the coefficients for PCR linearity (R2) and efficiency (E) were 0.997 and 0.80, respectively. We tested the performance of the hly-IAC Q-PCR assay using various broth media and food matrices. Fraser and half-Fraser media, raw pork, and raw or cold-smoked salmon were strongly PCR-inhibitory. This Q-PCR assay for L. monocytogenes, the first incorporating an IAC to be described for quantitative detection of a food-borne pathogen, is a simple and robust tool facilitating the identification of false negatives or underestimations of contamination loads due to PCR failure.

| S-EPMC1317324 | biostudies-literature

A novel specific duplex real-time RT-PCR method for absolute quantitation of Grapevine Pinot gris virus in plant material and single mites.

Project description:Grapevine Pinot gris virus (GPGV) is a widely distributed grapevine pathogen that has been associated to the grapevine leaf mottling and deformation disease. With the aim of better understanding the disease epidemiology and providing efficient control strategies a specific and quantitative duplex TaqMan real-time RT-PCR assay has been developed. This method has allowed reliable quantitation of the GPGV titer ranging from 30 up to 3 x 108 transcript copies, with a detection limit of 70 viral copies in plant material. The assay targets a grapevine internal control that reduces the occurrence of false negative results, thus increasing the diagnostic sensitivity of the technique. Viral isolates both associated and non-associated to symptoms from Greece, Slovakia and Spain have been successfully detected. The method has also been applied to the absolute quantitation of GPGV in its putative transmission vector Colomerus vitis. Moreover, the viral titer present in single mites has been determined. In addition, in the current study a new polymorphism in the GPGV genome responsible for a shorter movement protein has been found. A phylogenetic study based on this genomic region has shown a high variability among Spanish isolates and points to a different evolutionary origin of this new polymorphism. The methodology here developed opens new possibilities for basic and epidemiological studies as well as for the establishment of efficient control strategies.

| S-EPMC5953474 | biostudies-literature

Quantitation of rabbit cytokine mRNA by real-time RT-PCR.

Project description:Fundamental understanding of rabbit immunology and the use of the rabbit as a disease model have long been hindered by the lack of immunological assays specific to this species. In the present study, we sought to develop a method to quantitate cytokine expression in rabbit cells and tissues. We report the development of a quantitative real-time RT-PCR method for measuring the relative levels of rabbit IFN-gamma, IL-2, IL-4, IL-10 and TNF-alpha mRNA. Quantitation was accomplished by comparison to a standard curve generated using plasmid DNA containing partial sequences of the relevant cytokines. Experimental studies demonstrate applicability of this assay to quantitate cytokine mRNA levels from rabbit spleen cells following mitogen stimulation. We have further utilized this assay to also examine cytokine expression in rabbit tissues during experimental syphilis infection.

| S-EPMC2041851 | biostudies-literature

Simultaneous fitting of real-time PCR data with efficiency of amplification modeled as Gaussian function of target fluorescence.

Project description:BackgroundIn real-time PCR, it is necessary to consider the efficiency of amplification (EA) of amplicons in order to determine initial target levels properly. EAs can be deduced from standard curves, but these involve extra effort and cost and may yield invalid EAs. Alternatively, EA can be extracted from individual fluorescence curves. Unfortunately, this is not reliable enough.ResultsHere we introduce simultaneous non-linear fitting to determine - without standard curves - an optimal common EA for all samples of a group. In order to adjust EA as a function of target fluorescence, and still to describe fluorescence as a function of cycle number, we use an iterative algorithm that increases fluorescence cycle by cycle and thus simulates the PCR process. A Gauss peak function is used to model the decrease of EA with increasing amplicon accumulation. Our approach was validated experimentally with hydrolysis probe or SYBR green detection with dilution series of 5 different targets. It performed distinctly better in terms of accuracy than standard curve, DART-PCR, and LinRegPCR approaches. Based on reliable EAs, it was possible to detect that for some amplicons, extraordinary fluorescence (EA > 2.00) was generated with locked nucleic acid hydrolysis probes, but not with SYBR green.ConclusionIn comparison to previously reported approaches that are based on the separate analysis of each curve and on modelling EA as a function of cycle number, our approach yields more accurate and precise estimates of relative initial target levels.

| S-EPMC2276494 | biostudies-literature

A universal influenza A and B duplex real-time RT-PCR assay.

Project description:A high throughput universal influenza A and B duplex real-time RT-PCR was developed to meet effectively the heightened surveillance and diagnostic needs essential in managing influenza infections and outbreaks. Primers and probes, designed to target highly conserved regions of the matrix protein of influenza A and the nucleoprotein of influenza B, were optimized using the high-throughput LightCycler 480 II system. Analytical sensitivity and specificity were characterized using RNA transcripts diluted serially, archived non-influenza respiratory viruses, and proficiency test samples. Eighty-nine clinical samples were tested in parallel against existing influenza A and B monoplex assays. Once validated, the duplex assay was applied prospectively on 2,458 clinical specimens that were later subtyped. In April 2011, the emergence of an influenza B variant necessitated the inclusion of an additional modified probe for influenza B and revalidation of the revised protocol. The lower detection limits of the assay were 50 copies/PCR. There was no cross-reactivity against any non-influenza respiratory virus and all proficiency testing materials were identified correctly. The parallel testing revealed a 98.9% overall agreement. Routine application of the assay revealed high sensitivity and specificity for the detection of influenza A/H1N1/2009, A/H3N2 and influenza B. Assay C(q) values correlated well between the pre- and post-revision protocols for influenza A (r(2) = 0.998) and B (r(2) = 0.999). The revised protocol detected three additional novel influenza B variant cases in 200 specimens reported previously as influenza B negative. This in-house assay offers a highly sensitive and specific option for laboratories seeking to expand their influenza testing capacity.

| S-EPMC7166972 | biostudies-literature

Real-time RT-PCR for the detection and quantitation of Oropouche virus.

Project description:Oropouche virus (OROV) causes an acute, systemic febrile illness, and in certain regions of South America, this represents the second most common human arboviral infection after dengue virus. A new real-time RT-PCR was developed for OROV and reassortant species. The new OROV rRT-PCR proved linear across 6-7 orders of magnitude with a lower limit of 95% detection of 5.6-10.8 copies/?L. Upon testing dilutions of OROV and Iquitos virus reference genomic RNA, all dilutions with >10 copies/?L were detected in both the OROV rRT-PCR and a comparator molecular assay, but the OROV rRT-PCR detected more samples with ?10 copies/?L (8/14 vs 0/13, respectively, P?=?0.002). In a set of 100 acute-phase clinical samples from Paraguay patients with a suspected arboviral illness, no patients tested positive for OROV RNA using either assay. The OROV rRT-PCR provides a sensitive molecular assay for the study of this important yet neglected tropical arboviral infection.

| S-EPMC6906250 | biostudies-literature

A Novel Pretreatment-Free Duplex Chamber Digital PCR Detection System for the Absolute Quantitation of GMO Samples.

Project description:Digital polymerase chain reaction (PCR) has developed rapidly since it was first reported in the 1990s. However, pretreatments are often required during preparation for digital PCR, which can increase operation error. The single-plex amplification of both the target and reference genes may cause uncertainties due to the different reaction volumes and the matrix effect. In the current study, a quantitative detection system based on the pretreatment-free duplex chamber digital PCR was developed. The dynamic range, limit of quantitation (LOQ), sensitivity and specificity were evaluated taking the GA21 event as the experimental object. Moreover, to determine the factors that may influence the stability of the duplex system, we evaluated whether the pretreatments, the primary and secondary structures of the probes and the SNP effect influence the detection. The results showed that the LOQ was 0.5% and the sensitivity was 0.1%. We also found that genome digestion and single nucleotide polymorphism (SNP) sites affect the detection results, whereas the unspecific hybridization within different probes had little side effect. This indicated that the detection system was suited for both chamber-based and droplet-based digital PCR. In conclusion, we have provided a simple and flexible way of achieving absolute quantitation for genetically modified organism (GMO) genome samples using commercial digital PCR detection systems.

| S-EPMC4813257 | biostudies-literature

Real-time PCR assay using molecular beacon for quantitation of hepatitis B virus DNA.

Project description:Levels of hepatitis B virus (HBV) DNA in the blood serve as an important marker in monitoring the disease progression and treatment efficacy of chronic HBV infection. Several commercial assays are available for accurate measurement of HBV genomic DNA, but many of them are hampered by relatively low sensitivity and limited dynamic range. The aim of this study was to develop a sensitive and accurate assay for measuring HBV genomic DNA using real-time PCR with a molecular beacon (HBV beacon assay). The performance of this assay was validated by testing serial dilutions of the two EUROHEP HBV DNA standards (ad and ay subtypes) of known concentrations. The assay showed low intra-assay (<7%) and interassay (<5%) variations for both subtypes. Its dynamic range was found to be 10(1) to 10(7) copies per reaction (1.0 x 10(2) to 1.0 x 10(9) copies ml(-1)). The assay was further evaluated clinically using serum samples from 175 individuals with chronic hepatitis B. The HBV DNA level measured by this assay showed good correlation with that measured by the commercially available COBAS AMPLICOR HBV Monitor test (r = 0.901; P < 0.001). The higher sensitivity and broader dynamic range of this assay compared to the existing commercial assays will provide an ideal tool for monitoring disease progression and treatment efficacy in HBV-infected patients, in particular for those with low levels of HBV viremia.

| S-EPMC497581 | biostudies-other

Duplex real-time PCR assay for rapid detection of ampicillin-resistant Enterococcus faecium.

Project description:Rapid and accurate identification of carriers of resistant microorganisms is an important aspect of efficient infection control in hospitals. Traditional identification methods of antibiotic-resistant bacteria usually take at least 3 to 4 days after sampling. A duplex real-time PCR assay was developed for rapid detection of ampicillin-resistant Enterococcus faecium (ARE). Primers and probes that are used in this assay specifically detected the D-Ala-D-Ala ligase gene of E. faecium and the modified penicillin-binding protein 5 gene (pbp5) carrying the Glu-to-Val substitution at position 629 (Val-629) in a set of 129 tested E. faecium strains with known pbp5 sequence. Presence of the Val-629 in the strain set from 11 different countries was highly correlated with ampicillin resistance. In a screening of hospitalized patients, the real-time PCR assay yielded a sensitivity and a specificity for the detection of ARE colonization of 95% and 100%, respectively. The results were obtained 4 h after samples were harvested from overnight broth of rectal swab samples, identifying both species and the resistance marker mutation in pbp5. This novel assay reliably identifies ARE 2 to 3 days more quickly than traditional culture methods, thereby increasing laboratory throughput, making it useful for rectal screening of ARE. The assay demonstrates the advantages of real-time PCR for detection of nosocomial pathogens.

| S-EPMC321536 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data