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PubMed2001

A novel method to compensate for different amplification efficiencies between patient DNA samples in quantitative real-time PCR.

Quantification of residual disease by real-time polymerase chain reaction (PCR) will become a pivotal tool in the development of patient-directed therapy. In recent years, various protocols to quantify minimal residual disease in leukemia or lymphoma patients have been developed. These assays assume that PCR efficiencies are equal for all samples. Determining t(14;18) and albumin reaction efficiencies for sixteen follicular lymphoma patient samples revealed higher efficiencies for blood samples than for lymph node samples in general. However, within one sample both reactions had equivalent efficiencies. Differences in amplification efficiencies between patient samples (low efficiencies) and the calibrator in quantitative analyses result in the underestimation of residual disease in patient samples whereby the weakest positive patient samples are at highest error. Based on these findings for patient samples, the efficiency compensation control was developed. This control includes two reference reactions in a multiplex setting, specific for the beta-actin and albumin housekeeping genes that are present in a constant ratio within DNA templates. The difference in threshold cycle values for both reference reactions, ie, the Ct(2) value, is dependent on the amplification efficiency, and is used to compensate for efficiency differences between patient samples and the calibrator. The beta-actin reference reaction is also used to normalize for DNA input. Furthermore, the efficiency compensation control facilitates identification of patient samples that are so contaminated with PCR inhibitory compounds that different amplification reactions are affected to a different extent. Accurate quantitation of residual disease in these samples is therefore impossible with the current quantitative real-time PCR protocols. Identification and exclusion of these inadequate samples will be of utmost importance in quantitative retrospective studies, but even more so, in future molecular diagnostic analyses.

Actins↗
PubMed2004

Quantitation of mule duck in goose foie gras using TaqMan real-time Polymerase Chain Reaction.

A real-time quantitative Polymerase Chain Reaction (PCR) method has been developed for the quantitation of mule duck (Anas platyrhynchos x Cairina moschata) in binary duck/goose foie gras mixtures. The method combines the use of real-time PCR with duck-specific and endogenous control "duck + goose" primers to measure duck content and total foie gras content, respectively. Both PCR systems (duck-specific and duck + goose) were designed on the mitochondrial 12S ribosomal RNA gene (rRNA). The duck-specific system amplifies a 96 bp fragment from duck DNA, whereas the duck + goose system amplifies a 120 bp fragment from duck and goose DNA. The method measures PCR product accumulation through a FAM-labeled fluorogenic probe (TaqMan). The C(t) (threshold cycle) values obtained from the duck + goose system are used to normalize the ones obtained from the duck-specific system. Analysis of experimental duck/goose foie gras binary mixtures demonstrated the suitability of the assay for the detection and quantitation of duck in the range of 1-25%. This genetic marker can be very useful to avoid mislabeling or fraudulent species substitution of goose by duck in foie gras.

Animals↗
PubMed2005

Oncofoetal fibronectin--a tumour-specific marker in detecting minimal residual disease in differentiated thyroid carcinoma.

Supposedly, thyrocyte-specific transcripts such as thyroglobulin (Tg) and thyroid-stimulating hormone receptor (TSH-R) were proposed to be useful for the diagnosis of circulating tumour cells in patients suffering from differentiated thyroid carcinoma (DTC). However, several research groups reported blood-borne Tg transcripts in healthy individuals. This study determines in particular the origin of Tg mRNA in nucleated blood cells and analyses whether other tumour-associated sequences are absent in leukocytes, but widely expressed in DTC. Therefore, expression analyses for Tg, TSH-R, cytokeratin 19 (CK 19), human telomerase reverse transcriptase (hTERT) and oncofoetal fibronectin (onfFN) were carried out using cDNAs derived from (1) leukocyte fractions, (2) 18 follicular thyroid carcinomas (FTCs) and 48 papillary thyroid carcinomas (PTCs), and (3) leukocytes of two thyrocyte-depleted individuals treated for C-cell carcinoma of the thyroid. Expression of onfFN was additionally analysed by semiquantitative RT-PCR and by quantitative fluorescence-based real-time PCR. Tg and TSH-R expression was demonstrated not only in both athyroid individuals, but in all leukocyte subgroups tested, while hTERT was absent in resting CD4+ cells and only weakly expressed in the CD8+ group. CK 19 was notable in each leukocyte population except for resting CD14(+), as well as for activated and resting CD19+ cells. All blood cell fractions proved negative for onfFN mRNA, whereas its presence in thyroid carcinoma was 78/98% (FTC/PTC). Threshold cycle values were calculated at: porphobilinogen deaminase (PBGD) = 25.95+/-0.73 (FTC)/24.55+/-5.43 (PTC) (P = 0.2878); onfFN = 25.48+/-3.15 (FTC)/21.44+/-3.44 (PTC) (*P = 0.0001). Finally, onfFN transcripts were detected in blood samples of six out of nine patients with known DTC metastases, demonstrating a reliable assay functionality. We propose that real-time RT-PCR of onfFN mRNA is superior to other markers in monitoring minimal residual disease in DTC with regard to both assay sensitivity and specificity.

Adenocarcinoma, Follicular↗
PubMed2007

Evaluation of real-time PCR for the early detection of Legionella pneumophila DNA in serum samples.

Legionella pneumonia can be difficult to diagnose. Existing laboratory tests all have shortcomings, especially in the ability to diagnose Legionnaires' disease (LD) at an early stage of the disease in a specimen that is readily obtainable. The aim of this study was to assess the performance of PCR as a rapid diagnostic method and to compare the results of different PCR assays of serum samples from patients with LD. Samples included 151 serum samples from 68 patients with proven LD and 60 serum samples from 36 patients with respiratory tract infections other than Legionella. PCR assays were based on the 5S rRNA gene, 16S rRNA gene and the mip gene. The samples from patients with infections caused by pathogens other than Legionella all tested negative in PCR. Among the patients with proven LD 54.4 % (37/68) tested positive in 5S rRNA PCR, 52.9 % (36/68) in mip gene PCR and 30.9 % (21/68) in 16S rRNA PCR in the first available serum sample. The association between threshold cycle value in 5S PCR positive serum samples (n=49) and C-reactive protein value was determined, and showed a strong negative correlation (Pearson correlation coefficient r=-0.63, P<0.0001). In addition to existing tests for the diagnosis of LD, detection of Legionella DNA in serum could be a useful tool for early diagnosis of LD caused by any Legionella species and serogroup, and has the potential to provide a diagnosis in a time frame that could affect initial infection management.

Adult↗
PubMed2006

Sensitive quantitative detection of commensal bacteria by rRNA-targeted reverse transcription-PCR.

A sensitive rRNA-targeted reverse transcription-quantitative PCR (RT-qPCR) method was developed for exact and sensitive enumeration of subdominant bacterial populations. Using group- or species-specific primers for 16S or 23S rRNA, analytical curves were constructed for Escherichia coli, Enterococcus faecalis, Staphylococcus aureus, Clostridium perfringens, and Pseudomonas aeruginosa, and the threshold cycle value was found to be linear up to an RNA amount of 10(-3) cell per RT-PCR. The number of bacteria in culture was determined by RT-qPCR, and the results correlated well with the CFU count over the range from 10(0) to 10(5) CFU. The bacterial counts obtained by RT-qPCR were the same as the CFU counts irrespective of the growth phase in vitro, except for C. perfringens during starvation periods; the viable cell counts obtained by using a combination of 4',6-diamidino-2-phenylindole (DAPI) staining and SYTO9-propidium iodide double staining were in good agreement with the RT-qPCR counts rather than with the CFU counts. The RT-qPCR method could detect endogenous Enterobacteriaceae and P. aeruginosa in feces of hospitalized patients (n = 38) at a level of 10(3) cells per g of feces, and for enumeration of S. aureus or P. aeruginosa spiked into human peripheral blood, the lower detection limit for RT-qPCR quantification of the bacteria was 2 cells per ml of blood, suggesting that this method was equivalent to the conventional culture method. As only 5 h was needed for RT-qPCR quantification, we suggest that rRNA-targeted RT-qPCR assays provide a sensitive and convenient system for quantification of commensal bacteria and for examining their possible invasion of a host.

Adult↗
PubMed2004

Rapid detection of Vibrio vulnificus in shellfish and Gulf of Mexico water by real-time PCR.

In this paper we describe optimization of SYBR Green I-based real-time PCR parameters and testing of a large number of microbial species with vvh-specific oligonucleotide primers to establish a rapid, specific, and sensitive method for detection of Vibrio vulnificus in oyster tissue homogenate and Gulf of Mexico water (gulf water). Selected oligonucleotide primers for the vvh gene were tested for PCR amplification of a 205-bp DNA fragment with a melting temperature of approximately 87 degrees C for 84 clinical and environmental strains of V. vulnificus. No amplification was observed with other vibrios or nonvibrio strains with these primers. The minimum level of detection by the real-time PCR method was 1 pg of purified genomic DNA or 10(2) V. vulnificus cells in 1 g of unenriched oyster tissue homogenate or 10 ml of gulf water. It was possible to improve the level of detection to one V. vulnificus cell in samples that were enriched for 5 h. The standard curves prepared from the real-time PCR cycle threshold values revealed that there was a strong correlation between the number of cells in unenriched samples and the number of cells in enriched samples. Detection of a single cell of V. vulnificus in 1 g of enriched oyster tissue homogenate is in compliance with the recent Interstate Shellfish Sanitation Conference guidelines. The entire detection method, including sample processing, enrichment, and real-time PCR amplification, was completed within 8 h, making it a rapid single-day assay. Rapid and sensitive detection of V. vulnificus would ensure a steady supply of postharvest treated oysters to consumers, which should help decrease the number of illnesses or outbreaks caused by this pathogen.

Animals↗
PubMed2000

Real-time PCR for quantitative detection of Toxoplasma gondii.

The protozoan Toxoplasma gondii is one of the most common infectious pathogenic parasites and can cause severe medical complications in infants and immunocompromised individuals. We report here the development of a real-time PCR-based assay for the detection of T. gondii. Oligonucleotide primers and a fluorescence-labeled TaqMan probe were designed to amplify the T. gondii B1 gene. After 40 PCR cycles, the cycle threshold values (C(T)) indicative of the quantity of the target gene were determined. Typically, a C(T) of 25.09 was obtained with DNA from 500 tachyzoites of the T. gondii RH strain. The intra-assay coefficients of variation (CV) were 0.4, 0.16, 0.24, and 0.79% for the four sets of quadruplicate assays, with a mean interassay CV of 0.4%. These values indicate the reproducibility of this assay. Upon optimization of assay conditions, we were able to obtain a standard curve with a linear range (correlation coefficient = 0.9988) across at least 6 logs of DNA concentration. Hence, we were able to quantitatively detect as little as 0.05 T. gondii tachyzoite in an assay. When tested with 30 paraffin-embedded fetal tissue sections, 10 sections (33%) showed a C(T) of <40 and were scored as positive for this test. These results were consistent with those obtained through our nested-PCR control experiments. We have developed a rapid, sensitive, and quantitative real-time PCR for detection of T. gondii. The advantages of this technique for the diagnosis of toxoplasmosis in a clinical laboratory are discussed.

Animals↗
PubMed2003

Comparison of a new quantitative ompA-based real-Time PCR TaqMan assay for detection of Chlamydia pneumoniae DNA in respiratory specimens with four conventional PCR assays.

Chlamydia pneumoniae, an important respiratory pathogen, is difficult to culture, and detection rates by conventional PCRs vary considerably. A new quantitative ompA-based real-time PCR assay based on TaqMan technology for detection of C. pneumoniae in respiratory samples is described, and its performance in terms of sensitivity and reproducibility is compared with those of four published conventional PCRs (one single-step PCR targeting a cloned PstI fragment; two nested PCRs, one targeting the 16S rRNA gene followed by hybridization and the other targeting the ompA gene; and a touchdown enzyme time-release [TETR] PCR also targeting the 16S rRNA gene). Both ompA-based PCRs showed the best analytical sensitivity. All five assays could detect even lower target levels from spiked sputum, with the 16S rRNA assays performing better than the ompA-based nested PCR (10(-6) inclusion-forming units [IFU] were detected in four of four and two of four replicates by the 16S rRNA TETR PCR and the 16S rRNA nested PCR, respectively). In general, the ompA-based real-time protocol produced the most consistent positive results for all replicates tested down to 10(-6) IFU. Eight of 45 patient sputum specimens (18%) were C. pneumoniae DNA positive in at least one of four replicates tested by at least one assay. Without taking into consideration the analytical sensitivity or the reproducibility of the test results, the numbers of C. pneumoniae DNA-positive sputum specimens (n = 8) were four, three, two, two, and one for the 16S rRNA TETR assay, the PstI-based single-step PCR, the ompA-based real-time PCR, the ompA-based nested touchdown PCR, and the 16S rRNA-based nested PCR, respectively. However, the overall rate of concordance of positive results was low. Only one cell culture-positive sputum specimen was positive by four of five assays (14 of 16 replicates; mean cycle threshold value, 25; 10(8) particles/ml of sputum). Thirty-seven specimens were C. pneumoniae negative by all five assays for all replicates tested, as were all negative controls (n = 65 to 100 per testing panel). No PCR inhibitors were detected by real-time PCR or by the 16S rRNA-based nested assay. We confirm that the analytical sensitivity of an assay for the detection of C. pneumoniae does not necessarily predict its ability to detect its target in sputum. A quantitative, fast, and easy-to-handle diagnostic approach such as the ompA-based real-time TaqMan PCR described here might improve the detection of C. pneumoniae in respiratory samples.

Adult↗
PubMed2005

Multiplex real-time PCR assay using Scorpion probes and DNA capture for genotype-specific detection of Giardia lamblia on fecal samples.

Two major genotypic assemblages of Giardia lamblia infect humans; the epidemiologic significance of this phenomenon is poorly understood. We developed a single-vessel multiplex real-time PCR (qPCR) assay that genotypes Giardia infections into assemblages A and/or B directly from fecal samples. The assay utilized Scorpion probes that combined genotype-specific primers and probes for the 18S rRNA gene into the same molecule. The protocol was capable of detecting as few as 20 trophozoites per PCR on fecal DNA isolated using a commercial method or 1.25 trophozoites per PCR on fecal DNA isolated using a G. lamblia-specific oligonucleotide capture technique. The assay was specific for fecal specimens, with no amplification of the discordant genotype with the opposite Scorpion probe. When 97 clinical specimens from Bangladesh were used, the multiplex PCR assay detected 95% (21 of 22) of Giardia microscopy-positive specimens and 18% (13 of 74) of microscopy-negative specimens. Microscopy-negative and qPCR-positive specimens had higher average cycle threshold values than microscopy-positive and qPCR-positive specimens, suggesting that they represented true low-burden infections. Most (32 of 35) infections were assemblage B infections. This single-reaction multiplex qPCR assay distinguishes assemblage A Giardia infections from assemblage B infections directly on fecal samples and may aid epidemiologic investigation.

Animals↗
PubMed2005

Use of a novel virus inactivation method for a multicenter avian influenza real-time reverse transcriptase-polymerase chain reaction proficiency study.

Proficiency assessments are important elements in quality control for diagnostic laboratories. Traditionally, proficiency testing for polymerase chain reaction (PCR)-based assays has involved the use of clinical samples, samples "spiked" with live agents or DNA plasmids. Because of government regulations and biosecurity concerns, distribution of live high-consequence pathogens of livestock and poultry, such as avian influenza, is not possible, and DNA plasmids are not technically suitable for evaluating RNA virus detection. Therefore, a proficiency testing panel using whole avian influenza in a diluent containing a phenolic disinfectant that inactivates the virus while preserving the RNA for at least 8 weeks at -70 C was developed and used in a multicenter proficiency assessment for a type A influenza real-time reverse transcriptase (RT)-PCR test. The test, which was highly standardized, except for variation in the real-time RT-PCR equipment used, was shown to be highly reproducible by proficiency testing in 12 laboratories in the United States, Canada, and Hong Kong. Variation in cycle threshold values among 35 data sets and 490 samples was minimal (CV = 5.19%), and sample identifications were highly accurate (96.7% correct identifications) regardless of real-time PCR instrumentation.

Disinfectants↗
PubMed2006

Effect of probe-site mismatches on detection of virulent Newcastle disease viruses using a fusion-gene real-time reverse transcription polymerase chain reaction test.

Virulent forms of Newcastle disease virus (NDV) are a major concern for poultry producers around the world and the rapid diagnosis of an outbreak is crucial to any control program. A validated real-time reverse transcription-PCR test (fusion test) directed at the fusion-cleavage site of NDV was developed to differentiate virulent Newcastle disease virus strains from those of low virulence, however one virulent isolate, Dove/Italy/2736/2000, escaped detection during the initial evaluation of the test. The objectives of this study were to determine how this isolate differed from other detectable isolates, to identify other isolates that may fail detection, and to characterize the effect of specific probe-site mutations on the fusion test at a range of annealing temperatures. Using a virulent NDV isolate (Game fowl/US(CA)/2002) as a backbone that has 100% identity to the fusion-test probe, specific changes were made to the fusion-test probe-site to reflect the unique mismatches found in Dove/Italy/2736/2000 and other selected regions of the probe. Mutated clones with mismatches unique to Dove/Italy/2736/2000 at positions 6, 13, and 14 were not detected until annealing temperatures were lowered to 50 degrees C. Those detected at 58 degrees C contained 1-2 mismatches (position 1 and 6, 13 and 14, or 14 only) although increased cycle threshold values compared to the parent clone indicated decreased sensitivity. Data from this study predicts that the fusion test may fail to detect some viruses among lineage 4b and potential solutions to identify this subset of viruses include lowering the annealing temperature or modifying the probe.

Animals↗
PubMed2006

Freezing complete polymerase chain reaction master mix reagents for routine molecular diagnostics.

The potential of storing complete frozen real-time polymerase chain reactions (PCRs) and real-time reverse transcription PCRs (RT-PCRs), which require only thawing and the addition of template nucleic acid before PCR cycling, was examined. Master mixes containing all necessary reagents at working concentration (except template nucleic acid) were aliquoted into single-reaction volumes and stored at -70 degrees C for periods of up to 8 months. Reactions were removed from storage and nucleic acid template was added and amplified using different real-time PCR instruments. Threshold cycle values were used to monitor changes in assay performance after storage. Results for hybridization probe and TaqMan probe assays showed that freezing complete real-time PCR and RT-PCR reaction mixes was possible without deterioration in assay performance. This approach has advantages for routine molecular diagnostics in areas such as convenience, test consistency, quality control, and ease of use by nonspecialist staff.

Animals↗
PubMed2006

Development and application of a real-time quantitative PCR for prenatal detection of fetal alpha(0)-thalassemia from maternal plasma.

In order to provide a noninvasive prenatal diagnosis of alpha(0)-Thalassemia (Southeast Asian [SEA] deletion), we have developed a real-time quantitative semi-nested polymerase chain reaction (PCR) method for identifying the fetal alpha(0)-Thalassemia in maternal plasma. Analysis was performed using DNA extracted from 200 muL plasma from 13 pregnant women during 8-20 weeks of gestation who carried fetuses with normal (2), alpha(0)-Thalassemia carrier (8), Hb H disease (1), and homozygous alpha(0)-Thalassemia (Hb Bart's hydrops fetalis (2). The alpha(0)-Thalassemia was detected using a two-step PCR. Plasma DNA was amplified conventionally using alpha(0)-Thalassemia-specific primers and a portion of the first PCR product was subjected to a semi-nested real-time q-PCR using the SYBR green I chemistry for fluorescence detection. Calibration curve for alpha(0)-Thalassemia quantification was prepared by assaying serial dilution of genomic DNA of an alpha(0)-Thalassemia carrier. Differences in the C(T) (threshold cycle) values and calculated concentrations of amplified DNA among normal fetus, alpha(0)-Thalassemia carrier, Hb H disease, and homozygous alpha(0)-Thalassemia were clearly observed, which could help in prenatal prediction of the fetal genotype. This noninvasive prenatal detection of alpha(0)-Thalassemia in maternal plasma should enhance prenatal diagnostic options for this common genetic disorder in routine DNA diagnostic setting.

DNA↗
PubMed2006

Application of a real-time PCR assay to detect and quantify the myxozoan parasite Ceratomyxa shasta in river water samples.

Ceratomyxa shasta is a virulent pathogen of salmonid fishes that is enzootic in the Pacific Northwest of North America. Current parasite detection methods involve sentinel fish exposures that are laborious and time-consuming. As a substitute, a filtering protocol and a quantitative real-time TaqMan polymerase chain reaction (QPCR) assay were developed to detect and enumerate parasite spores in river water. Fluorescence was detected from both the myxospore and actinospore stages of the parasite but not from the fish or polychaete hosts or from 9 other myxozoans tested. Less than 1/1000th of a spore was detected, indicating each had >1000 copies of the target 18S rRNA gene. The assay detected 1 spore in 1 l river water. Inhibition of the assay by some river samples was overcome by reducing the template volume and including bovine serum albumin in the reaction; occasionally a second purification step was required. The QPCR methodology was utilised to investigate the temporal and spatial distribution of C. shasta in the Klamath River, Oregon/ California. The parasite was detected throughout the river, and 2 of 5 tributaries tested contributed parasites to the mainstem. Correlation of QPCR cycle threshold values with a standard curve for known starting numbers of whole spores revealed several sites where parasite abundance was in excess of 20 spores l(-1). Although QPCR data corroborated results of sentinel fish exposures, spore numbers did not correlate consistently with mortality in the exposure groups. The water sampling and filtering protocol combined with the QPCR assay is a simple and relatively rapid method for detection and quantification of parasite levels in environmental water samples.

Animals↗
PubMed2026

Molecular epidemiological characteristics of H9N2 subtype avian influenza virus in the external environment of western Zhejiang, China, 2014-2025.

OBJECTIVE: To elucidate the epidemiological distribution patterns of avian influenza virus (AIV) in the external environment of western Zhejiang from 2014 to 2025, analyze the molecular epidemiological characteristics of the H9N2 subtype, and assess its public health risks. METHODS: According to the Zhejiang Provincial Surveillance Program for Avian Influenza in Occupationally Exposed Populations and External Environments, real-time RT-PCR was used to detect AIV subtypes in environmental specimens. H9N2-positive samples with cycle threshold values <30 were inoculated into specific pathogen-free (SPF) embryonated chicken eggs for virus isolation, followed by whole-genome sequencing and bioinformatics analysis for phylogenetic and molecular characterization. RESULTS: A total of 7,762 specimens were tested from 2014 to 2025, with an overall positivity rate of 34.64% (2,689/7,762) for AIV. Significant differences in positivity rates were observed in seasons, regions, sampling sites, and specimen types (all p&#x202f;<&#x202f;0.001). AIV activity peaked in winter and spring, with the highest rates detected in live poultry markets and chopping board swabs. The H9 was the predominant subtype, with co-circulation of multiple subtypes. All 48 H9N2 subtype isolates belonged to the G57 genotype, with the hemagglutinin (HA) and neuraminidase (NA) genes falling into the Y280-like branch, while the internal genes exhibited a mosaic pattern combining G1-like and F/98-like lineages. Molecular characterization analysis revealed multiple mammalian adaptive mutations, involving alterations in receptor-binding sites (T163N, H191N, T197D, T198V, Q234L, Q235M), antigenic epitopes (D280G, N285S), and glycosylation sites (218NRTF, 313NCSK). NA stalk deletion (62-64 aa), along with multiple mutations in the hemadsorption site (E/K368N, D369S/G, D401G/V, N402D, W403L/R, Q432H). Additionally, multiple key amino acid substitutions were also identified in the internal proteins. CONCLUSION: The external environment in western Zhejiang exhibits a high prevalence of AIVs with pronounced spatiotemporal clustering. H9 was the dominant subtype and co-circulated with multiple subtypes, with live poultry markets and slaughterhouses identified as high-risk settings. The H9N2 subtype AIV has accumulated multiple mammalian adaptive mutations, and exhibits genetic linkages across eastern Chinese provinces. These findings collectively underscore the need for an integrated One Health surveillance and early-warning system to reduce the risk of human infections with avian influenza.

Influenza in Birds↗
PubMed2006

Multiplex real-time PCR detection of heat-labile and heat-stable toxin genes in enterotoxigenic Escherichia coli.

A multiplex real-time PCR method was developed for detection of heat-labile and heat-stable toxin genes in enterotoxigenic Escherichia coli. Approximately 10 CFU per reaction mixture could be detected in rinsates from produce samples. Several foods representative of varieties previously shown to have caused enterotoxigenic E. coli outbreaks were spiked and enriched for 4 or 6 h. Both heat-labile and heat-stable toxin genes could be detected in the foods tested, with the exception of hot sauce, with threshold cycle values ranging from 25.2 to 41.1. A procedure using membrane filtration which would allow enumeration of the enterotoxigenic E. coli population in a food sample in less than 28 h by real-time PCR analysis of colonies picked from media highly selective for E. coli was also developed.

Amino Acid Sequence↗
PubMed2000

Influence of DNA target melting behavior on real-time PCR quantification.

BACKGROUND: Quantitative real-time PCR is increasingly used to quantify copy numbers of nucleic acids for clinical applications. We observed that the measurements of allele imbalances of the tumor suppressor gene p16 and the oncogene ErbB-2 yielded results with variable precision under certain experimental conditions. METHODS: We used the LightCycler(TM) real-time PCR system to quantify different genomic target sequences using hybridization probes or SYBR Green for detection. RESULTS: With two primer/template systems (p16 and ErbB-2), we observed sinusoidal scattering of the threshold cycle values depending on the capillary position in the thermostated reaction chamber. This scattering depended on the denaturation temperature only when complete genomic DNA was used as template and did not occur when PCR product or restricted or boiled genomic DNA was used or the denaturation temperature in the first cycles was increased (and other targets, such as p53, HBB, IGF-1, GAPDH, and PBGD, did not show this behavior). CONCLUSIONS: Before a primer system is used for precise quantitative real-time PCR, the dependence of the quantification results on the positions of reaction tubes in the thermocycler should be tested. Our data indicate that amplification efficiencies, especially in the first cycles, depend not only on the priming efficiencies of the primers and the melting temperature of the amplicon, but also on the melting behavior of the amplicon's genomic vicinity. Complete denaturation of genomic DNA is necessary to maximize precision of quantitative PCR. Higher denaturation temperatures in the initial cycles or boiling of DNA before the PCR can improve the accuracy of quantification in some cases.

Cyclin-Dependent Kinase Inhibitor p16↗
PubMed2001

Simultaneous absolute quantification of target and control templates by real-time fluorescence reverse transcription-PCR using 4-(4'-dimethylaminophenylazo)benzoic acid as a dark quencher dye.

BACKGROUND: Despite the many advantages of real-time fluorescence reverse transcription-PCR (RT-PCR) as a quantitative analytical tool, simultaneous quantification of target and reference templates within one reaction has not been reported. We developed such an assay with an internal reference template. METHODS: For quantification of target and reference sequences, we used two fluorescent probes in one reaction vessel on an ABI PRISM 7700 SDS instrument. Fluorescent probes were labeled with either 6-carboxy-fluorescein or hexachloro-6-carboxy-fluorescein as reporter dye and 4-(4'-dimethylaminophenylazo)benzoic acid (DABCYL) as a dark quencher fluorophore. To test the sensitivity and specificity of this assay, serial dilutions of reference and target templates were analyzed in one PCR reaction. In the presence of 10 beta-actin molecules as control templates, 10(5) bcr/abl molecules were amplified, and 10(5) beta-actin molecules were amplified in the presence of 10 bcr/abl copies. We also performed single and duplex measurements on samples from five patients with documented Philadelphia chromosome-positive chronic myelogenous leukemia disease courses (72 samples) and three with minor bcr/abl+ acute myelogenous leukemias (26 samples). RESULTS: For M-bcr/abl duplex RT-PCR, the correlation coefficient (r) for starting template amounts and threshold cycle values was 0.99; for m-bcr/abl, r = 0.96, indicating a precise log-linear relation for 10-10(5) copies/100 ng of cDNA. In the same PCR reactions, r = 0.99 for beta-actin (coamplified with M-bcr/abl or m-bcr/abl) for 10(3)-10(7) copies/100 ng cDNA. The linear correlation coefficient for single and duplex measurements was 0.98 for M- and m-bcr/abl in patient samples. CONCLUSIONS: DABCYL can be used as dark quencher fluorophore in real-time fluorescence PCR. The duplex fluorescence RT-PCR assay for bcr/abl and beta-actin transcripts allows monitoring of bcr/abl+ leukemias.

Fluorescent Dyes↗