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Evaluation of quantitative PCR and branched-chain DNA assay for detection of hepatitis B virus DNA in sera from hepatocellular carcinoma and liver transplant patients.

This study evaluated the applicability of quantitative PCR (Q-PCR) and branched-chain DNA assays for detection of hepatitis B virus (HBV) DNA in sera. For 42 samples, the detection rates were 81 and 41%, respectively, with a correlation coefficient of 0.633. The Q-PCR is useful for early monitoring of HBV load in high-risk patients.

Adult↗

Quantitative PCR.

The classic molecular biology methods like Northern or Southern blot analyse non-amplified DNA or RNA, but need large amounts of nucleic acids, in many instances from tissues or cells that are heterogeneous. In contrast, polymerase chain reaction (PCR)-based techniques allow us to obtain genetic information through the specific amplification of nucleic acid sequences starting with a very low number of target copies. These reactions are characterized by a logarithmic amplification of the target sequences i.e. increase of PCR copies followed by a plateau phase showing a rapid decrease to zero of copy number increment per cycle. Accordingly, the amount of specific DNA product at the end of the PCR run bears no correlation to the number of target copies present in the original specimen. However, many applications in medicine or research require quantification of the number of specific targets in the specimen. This has generated a rapidly increasing need for the development of quantitative PCR techniques. Prominent examples are the determination of viral load in blood specimens for the diagnosis of HIV or HCV infections, the determination of changes in gene dosage through amplification or deletion e.g. of MDR-1, erb-B2, c-myc or the loss of heterozygosity in general. Finally, the analysis of gene expression on the mRNA level does require quantitative approaches to reverse transcriptase PCR, e.g. for studies in morphogenesis or the profiling of cancer cells. Recent advances in technology allow detection of the increment per cycle of a specifically generated PCR product in "real-time mode". Together with the new powerful methods to dissect heterogeneous tissues or fractionate bodily fluids, this now sets the stage for a detailed analysis not only of the genes and genetic changes within a single cell, but also of the use such cell makes of its genes e.g. in pharmacogenomics. Examples of recent developments of the technology and their applications will be given.

Gene Amplification↗

Abundance and distribution of Ostreococcus sp. in the San Pedro Channel, California, as revealed by quantitative PCR.

Ostreococcus is a genus of widely distributed marine phytoplankton which are picoplanktonic in size (<2 mum) and capable of rapid growth. Although Ostreococcus has been detected around the world, little quantitative information exists on its contribution to planktonic communities. We designed and implemented a genus-specific TaqMan-based quantitative PCR (qPCR) assay to investigate the dynamics and ecology of Ostreococcus at the USC Microbial Observatory (eastern North Pacific). Samples were collected from 5 m and the deep chlorophyll maximum (DCM) between September 2000 and August 2002. Ostreococcus abundance at 5 m was generally <5.0 x 10(3) cells ml(-1), with a maximum of 8.2 x 10(4) cells ml(-1). Ostreococcus abundance was typically higher at the DCM, with a maximum of 3.2 x 10(5) cells ml(-1). The vertical distribution of Ostreococcus was examined in March 2005 and compared to the distribution of phototrophic picoeukaryotes (PPE) measured by flow cytometry. The largest contribution to PPE abundance by Ostreococcus was approximately 70% and occurred at 30 m, near the DCM. Despite its relatively low abundance, the depth-integrated standing stock of Ostreococcus in March 2005 was approximately 30 mg C m(-2). Our work provides a new technique for quantifying the abundance of Ostreococcus and demonstrates the seasonal dynamics of this genus and its contribution to picoeukaryote biomass at our coastal sampling station.

Animals↗

Rapid Staphylococcus aureus agr type determination by a novel multiplex real-time quantitative PCR assay.

The accessory gene regulator (agr) is a crucial regulatory component of Staphylococcus aureus involved in the control of bacterial virulence factor expression. We developed a real-time multiplex quantitative PCR assay for the rapid determination of S. aureus agr type. This assay represents a rapid and affordable alternative to sequence-based strategies for assessing relevant epidemiological information.

Bacterial Proteins↗

A new quantitative PCR multiplex assay for rapid analysis of chromosome 17p11.2-12 duplications and deletions leading to HMSN/HNPP.

A 1.4-Mb tandem duplication, including the gene for peripheral myelin protein 22 (PMP22) in chromosome 17p11.2-12 is responsible for 70% of the cases of the demyelinating type 1 of Charcot-Marie-Tooth disease or hereditary motor and sensory neuropathy I (CMT1A/HMSN I). A reciprocal deletion of this CMT1A region causes the hereditary neuropathy with liability to pressure palsies (HNPP). The CMT1A duplication increases the PMP22 gene dosage from two to three, the HNPP deletion reduces the gene dosage from two to one. Currently, routine diagnosis of HMSN/HNPP patients is mainly performed with polymorphic markers in-between the repetitive elements flanking the CMT1A region. These show quantitative and/or qualitative changes in case of a CMT1A duplication and a homozygous allele pattern in case of HNPP deletion. In HNPP patients the deletion is usually confirmed by fluorescence in situ hybridisation (FISH). We now developed a reliable, single tube real-time quantitative PCR assay for rapid determination of PMP22 gene dosage directly. This method involves a multiplex reaction using FAM labelled Taqman-probe with TAMRA quencher derived from PMP22 exon 3 and a VIC labelled probe with non-fluorescent quencher from exon 12 of the albumin gene as internal reference. Copy number of the PMP22 gene was determined by the comparative threshold cycle method (deltadeltaCt). Each sample was run in quadruplicate and analysed at two different threshold levels. The level giving the smallest standard deviation was scored. We evaluated this method through the retrospective analysis of 252 HMSN patients with known genotype and could confirm the previous findings in 99% of cases. Two patients were wrongly diagnosed with microsatellite analysis while quantitative real-time PCR identified the correct genotype, as confirmed by FISH. Thus, this method shows superior sensitivity to microsatellite analysis and has the additional advantage of being a fast and uniform assay for quantitative analysis of both CMT1A and HNPP.

Biological Assay↗

Detection and quantification of phnE gene from oil-contaminated soil samples by competitive quantitative PCR.

The phnE gene encoding catechol 2,3-dioxygenase belonging to the meta-cleavage pathway was selected as the marker gene and was detected and quantified from soil samples by competitive quantitative PCR. A PCR primer pair was designed based on the phnE gene to amplify the target DNA bands and competitor DNA bands. The phnE gene was detected in two samples of three. In samples S1 and S2, the phnE gene copy number was 6.2 x 10(7)/g soil and 5.8 x 10(7)/g soil, respectively. But no phnE gene was detected in sample S3. The target DNA bands were extracted and expressed. The results confirmed that the target DNA bands were the native phnE genes.

Bacterial Proteins↗

A comparison between microsatellite and quantitative PCR analyses to detect frequent p16 copy number changes in primary bladder tumors.

We tested 70 primary bladder tumors for altered copy number of p16 (D9S1752) by microsatellite analysis and by a quantitative PCR (QPCR) assay. These two approaches were fully concordant for 53 tumors, including all 39 tumors in which microsatellite analysis detected loss. In addition, the QPCR method detected useful anomalies in 17 additional cases, including those in which D9S1752 was uninformative. QPCR was abnormal in 56 of 70 (80%) cases, whereas microsatellite analysis was abnormal in 39 of 70 (56%) cases. Although QPCR uses more DNA than microsatellite analysis, it represents a rapid, informative technique that can readily detect both chromosome 9p21 deletions and amplifications in primary bladder tumors without the need for electrophoretic separation.

DNA, Neoplasm↗

Quantitative-PCR assessment of Cryptosporidium parvum cell culture infection.

A quantitative TaqMan PCR method was developed for assessing the Cryptosporidium parvum infection of in vitro cultivated human ileocecal adenocarcinoma (HCT-8) cell cultures. This method, termed cell culture quantitative sequence detection (CC-QSD), has numerous applications, several of which are presented. CC-QSD was used to investigate parasite infection in cell culture over time, the effects of oocyst treatment on infectivity and infectivity assessment of different C. parvum isolates. CC-QSD revealed that cell culture infection at 24 and 48 h postinoculation was approximately 20 and 60%, respectively, of the endpoint 72-h postinoculation infection. Evaluation of three different lots of C. parvum Iowa isolate oocysts revealed that the mean infection of 0.1 N HCl-treated oocysts was only 36% of the infection obtained with oocysts treated with acidified Hanks' balanced salt solution containing 1% trypsin. CC-QSD comparison of the C. parvum Iowa and TAMU isolates revealed significantly higher levels of infection for the TAMU isolate, which agrees with and supports previous human, animal, and cell culture studies. CC-QSD has the potential to aid in the optimization of Cryptosporidium cell culture methods and facilitate quantitative evaluation of cell culture infectivity experiments.

Animals↗

Effects of temperature and salinity on Vibrio vulnificus population dynamics as assessed by quantitative PCR.

The abundance of Vibrio vulnificus in coastal environments has been linked to water temperature, while its relationship to salinity is less clear. We have developed a culture-independent, most-probable-number quantitative PCR approach to examine V. vulnificus population dynamics in Barnegat Bay, N.J. Based on the combined analysis of our results from Barnegat Bay and from the literature, the present data show that (i) V. vulnificus population dynamics are strongly correlated to water temperature and (ii) although the general trend is for V. vulnificus abundance to be inversely correlated with salinity, this relationship depends on salinity levels. Irrespective of temperature, high abundances of V. vulnificus are observed at 5 to 10 ppt, which thus appears to be the optimal salinity regime for their survival. At 20 to 25 ppt, V. vulnificus abundances show a positive correlation to salinity. Unsuccessful attempts to resuscitate V. vulnificus, combined with our inability to detect cells during the winter despite an assay adapted to detect viable but nonculturable (VBNC) cells, suggest that the decline and eventual disappearance of V. vulnificus from the water column during the winter months is due primarily to a significant reduction in population size and is not only the consequence of cells entering the VBNC state. These findings are in line with the hypothesis that the sediment serves as a refuge for a subpopulation of V. vulnificus over the winter and weather-driven mixing events during the spring initiate a summer bloom in the water column.

Chlorophyll↗

A real-time quantitative PCR comparative study between rat optic and sciatic nerves: determination of neuregulin-1 mRNA levels.

Injured axons from peripheral nervous system (PNS) possess the ability to regenerate. In contrast, regeneration of injured axons does not occur in the central nervous system (CNS) or occurs to a limited extent. Previous works have shown that rat sciatic nerve conditioned medium (CM) produced PC12 cells neuronal-like differentiation and neurite outgrowth. In the present work, we compared the expression of neuregulin-1s (NRG-1s) from rat sciatic and optic nerves as members of the PNS and CNS, respectively. Sciatic nerve CM showed a higher neurotrophic activity on PC12 cells than rat optic nerve CM. RT-PCR analysis verified the presence of all three types of NRG-1 mRNAs and their receptors in both types of nerves. Real-time quantitative PCR (QPCR) assays showed that the relative expression levels of all three types of NRG-1 mRNAs were higher in optic nerves than in sciatic nerves. Eleven-day cultured optic nerves showed an increased in NDF and SMDF when compared to freshly isolated optic nerves, whereas GGF decreased. However, 11-day-cultured sciatic nerves only showed an increase in SMDF mRNA. Western blots corroborated the differences in NRG-1 expression profile for both types of nerves and their CMs. Incubation of both CMs with the anti-pan-NRG-1 antibody showed that the neurotrophic activity of the optic nerve CM increased, whereas the sciatic nerve CM remained unchanged. These results indicated that different NRG-1 levels are expressed upon nerve degeneration and the balance between those levels and other neurotrophic factors could have an important role on nerve regeneration.

Animals↗

Quantitative PCR with colorimetric detection.

We describe a quantitative polymerase chain reaction (PCR) technique with a colorimetric read-out, which enables quantitation of various sequences on a single microplate within one day. Using the quantitation of human IL10 and beta-actin transcripts, we show that this technique provides better reproducibility, as results are derived from a series of PCRs made with various amounts of standard. This also enables the control of the consistency of the data and elimination of some artifactual results.

Actins↗

Quantitative PCR in the diagnosis of CMV infection and in the monitoring of viral load during the antiviral treatment in renal transplant patients.

Cytomegalovirus (CMV) infection is a significant problem in transplantation. In this study, a quantitative PCR test was compared with the CMVpp65 antigenemia assay not only in the diagnosis CMV infections but especially in the monitoring of viral loads during ganciclovir treatment of CMV disease in individual renal transplant patients. Altogether 342 blood specimens were obtained from 116 patients. Blood specimens were used for Cobas Amplicor Monitor plasma PCR and for the pp65 assay. Also shell vial culture was performed. The patients with a positive pp65 finding were monitored for CMV weekly during ganciclovir treatment and/or until the antigenemia subsided. CMV was detected in 31/116 (27%) patients, of whom 14 (12%) developed CMV disease and were treated with ganciclovir. CMV was found by shell vial culture in 13/14 cases, but by PCR and pp65 test in all 14 patients. CMV was detected in 156 (45%) samples; by PCR in 121/156 (range 344-103,000 copies/ml) and by pp65 test in 138/156 (range 1-1,000 positive cells/50,000 leukocytes) and by culture in 59/156 (38%) only. The peak viral loads were significantly (P<0.0001) higher in CMV disease than in untreated infections (19,650 vs. 379 copies/ml, and 100 vs. 5pp65 positive cells). In the monitoring of individual patients, the time-related CMV-DNAemia and pp65 antigenemia correlated well during the treatment of CMV disease. In conclusion, Cobas Amplicor Monitor plasma PCR and CMVpp65 antigen assays can be equally used in the diagnosis CMV infection and in the monitoring of viral load during antiviral treatment.

Antigens, Viral↗

Development of a quantitative PCR assay for monitoring HCV viraemia levels in patients with chronic hepatitis C.

The need to improve the efficacy of anti-viral agents directed against the hepatitis C virus (HCV) has prompted the development of quantitative tests to monitor viraemia levels in relation to therapy. In this respect, we have produced a non-competitive, quantitative PCR assay (Amplicor HCV Monitor) which is based on the single, combined, reverse transcription and amplification of the 5' non-coding region of HCV RNA as well as of an internal standard which serves to assess the overall efficiency of the system. The dynamic range of the assay and its precision allows for an accurate quantification between 1000 and 1,000,000 copies of the viral genome per ml. The results of the first clinical evaluation of the test indicated that determination of viraemia may have predictive value when assessed prior to treatment, as patients with less than 50,000 HCV RNA copies per ml tend to show a long-term response to interferon-alpha (IFN-alpha) treatment. Moreover, a decrease of more than 1.5-2 logarithms occurring at 1 month post-initiation of therapy predicts response in a more timely and accurate fashion than ALT measurement. This may help in a better selection of patients to treat, therapy approaches and schedules and in the optimization of the cost-benefit balance.

Chronic Disease↗

Detection of tumor-derived DNA in exfoliated cells of urine sediments with a novel quantitative PCR method based on allele-specific PCR.

We have established a novel quantitative method based on the allele-specific PCR, which uses the linearly amplified fragment of the PCR products as the internal control. The improved characteristics of the procedure are the high sensitivity for quantitation of the mutant alleles at ratios of up to 1:10000 and the reduced necessity of the optimization of the PCR conditions for each mutation. Using this modified allele-specific PCR, we could quantify the tumor alleles in the urine sediments of three patients with urothelial cancers that harbored different p53 gene mutations. This method can be applied to other genetic targets that have other types of alterations, such as deletions or insertions.

Alleles↗

Quantitative PCR analysis of two molluscan metallothionein genes unveils differential expression and regulation.

The mRNA levels of two components of the mussel (Mytilus galloprovincialis) metallothionein (MT) gene families, MT10 and MT20, were evaluated using real-time quantitative-PCR and Sybr Green I chemistry in animals exposed to heavy metals in vivo and in primary cell cultures. This method was highly specific in detecting the expression of the two genes over a widely dynamic range of starting DNA amounts, showing that the basal level of MT expression is mostly due to MT10 mRNA. Basal MT expression reflected the intracellular concentration of heavy metal as indicated by the use of the heavy metal chelator TPEN on primary cells. MT10 was observed to be inducible by Cd, Zn, and Cu ions, and to a lesser extent by Hg. By contrast, the MT20 expression level was very low under basal conditions, while its mRNA increased dramatically in response to Cd exposure, and to a lesser extend to Hg, leading to levels of expression similar to those of the MT10 gene. The essential metals Cu and Zn had a very small effect on the MT20 gene, whereas the concomitant exposure to Cu and H(2)O(2) produced a rapid rise of expression. In summary, data indicate that the MT isogenes are differentially regulated by heavy metals, while hydroxyl radicals may have a role in MT20 gene activation. Also, protein expression showed metal inducibility only after Cd exposure, suggesting the occurrence of posttranscriptional control mechanisms.

Animals↗

Prognostic value of CCND1 gene status in sporadic breast tumours, as determined by real-time quantitative PCR assays.

The CCND1 gene, a key cell-cycle regulator, is often altered in breast cancer, but the mechanisms underlying CCND1 dysregulation and the clinical significance of CCND1 status are unclear. We used real-time quantitative PCR and RT-PCR assays based on fluorescent TaqMan methodology to quantify CCND1 gene amplification and expression in a large series of breast tumours. CCND1 overexpression was observed in 44 (32.8%) of 134 breast tumour RNAs, ranging from 3.3 to 43.7 times the level in normal breast tissues, and correlated significantly with positive oestrogen receptor status (P=0.0003). CCND1 overexpression requires oestrogen receptor integrity and is exacerbated by amplification at 11q13 (the site of the CCND1 gene), owing to an additional gene dosage effect. Our results challenge CCND1 gene as the main 11q13 amplicon selector. The relapse-free survival time of patients with CCND1-amplified tumours was shorter than that of patients without CCND1 alterations, while that of patients with CCND1-unamplified-overexpressed tumours was longer (P=0.011). Only the good prognostic significance of CCND1-unamplified-overexpression status persisted in Cox multivariate regression analysis. This study confirms that CCND1 is an ER-responsive or ER-coactivator gene in breast cancer, and points to the CCND1 gene as a putative molecular marker predictive of hormone responsiveness in breast cancer. Moreover, CCND1 amplification status dichotomizes the CCND1-overexpressing tumors into two groups with opposite outcomes.

Adult↗

Detection of minimal residual disease in hematologic malignancies by real-time quantitative PCR: principles, approaches, and laboratory aspects.

Detection of minimal residual disease (MRD) has prognostic value in many hematologic malignancies, including acute lymphoblastic leukemia, acute myeloid leukemia, chronic myeloid leukemia, non-Hodgkin's lymphoma, and multiple myeloma. Quantitative MRD data can be obtained with real-time quantitative PCR (RQ-PCR) analysis of immunoglobulin and T-cell receptor gene rearrangements, breakpoint fusion regions of chromosome aberrations, fusion-gene transcripts, aberrant genes, or aberrantly expressed genes, their application being dependent on the type of disease. RQ-PCR analysis can be performed with SYBR Green I, hydrolysis (TaqMan) probes, or hybridization (LightCycler) probes, as detection system in several RQ-PCR instruments. Dependent on the type of MRD-PCR target, different types of oligonucleotides can be used for specific detection, such as an allele-specific oligonucleotide (ASO) probe, an ASO forward primer, an ASO reverse primer, or germline probe and primers. To assess the quantity and quality of the RNA/DNA, one or more control genes must be included. Finally, the interpretation of RQ-PCR MRD data needs standardized criteria and reporting of MRD data needs international uniformity. Several European networks have now been established and common guidelines for data analysis and for reporting of MRD data are being developed. These networks also include standardization of technology as well as regular quality control rounds, both being essential for the introduction of RQ-PCR-based MRD detection in multicenter clinical treatment protocols.

Adult↗

Detection of bacterial pathogens in municipal wastewater using an oligonucleotide microarray and real-time quantitative PCR.

As a first step toward building a comprehensive microarray, two low density DNA microarrays were constructed and evaluated for the accurate detection of wastewater pathogens. The first one involved the direct hybridization of wastewater microbial genomic DNA to the functional gene probes while the second involved PCR amplification of 23S ribosomal DNA. The genomic DNA microarray employed 10 functional genes as detection targets. Sensitivity of the microarray was determined to be approximately 1.0 microg of Esherichia coli genomic DNA, or 2 x 10(8) copies of the target gene, and only E. coli DNA was detected with the microarray assay using municipal raw sewage. Sensitivity of the microarray was enhanced approximately by 6 orders of magnitude when the target 23S rRNA gene sequences were PCR amplified with a novel universal primer set and allowed hybridization to 24 species-specific oligonucleotide probes. The minimum detection limit was estimated to be about 100 fg of E. coli genomic DNA or 1.4 x 10(2) copies of the 23S rRNA gene. The PCR amplified DNA microarray successfully detected multiple bacterial pathogens in wastewater. As a parallel study to verify efficiency of the DNA microarray, a real-time quantitative PCR assay was also developed based on the fluorescent TaqMan probes (Applied Biosystems).

Bacteria↗