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PubMed2005

Utility of Amsel criteria, Nugent score, and quantitative PCR for Gardnerella vaginalis, Mycoplasma hominis, and Lactobacillus spp. for diagnosis of bacterial vaginosis in human immunodeficiency virus-infected women.

Bacterial vaginosis (BV) is a clinical syndrome presenting with a malodorous vaginal discharge and increased vaginal pH. Diagnosis has been based on clinical Amsel criteria and direct Gram stain of vaginal secretions. Human immunodeficiency virus (HIV)-infected participants in the Women's Interagency HIV Study contributed cervicovaginal lavage (CVL) samples. Lactobacilli, Gardnerella vaginalis, and Mycoplasma hominis in cervicovaginal lavage samples were quantified by PCR. Gynecologic evaluation included Nugent score and Amsel criterion assessment. We compared the gold standard Nugent score to Amsel criteria and quantitative bacterial PCR for diagnosing BV in 203 CVL samples from women with Nugent scores of 7 to 10 (BV group) and 203 samples from women with BV Nugent scores of 0 to 3 ("No-BV" group). Only 75 of the 203 CVL samples from women with Nugent scores of 7 to 10 met positive Amsel criteria. Increasing levels of G. vaginalis and M. hominis and decreasing levels of lactobacilli were significantly associated with BV by Nugent score. Of the group with Nugent scores of 7 to 10, 83% and 81% had log(10) G. vaginalis counts and log(10) M. hominis counts greater than 6.81 and 4.82, respectively, while only 30% and 31% of the group with Nugent scores of 0 to 3 were above these thresholds, respectively. There was significant overlap in the log(10) lactobacillus counts between the two groups. Utilizing all three log(10) bacterial counts (G. vaginalis, M. hominis, and lactobacilli) in our model improved the sensitivity and specificity to 83% and 78%, respectively, in comparison with Nugent score. In this cohort, Amsel criteria were poorly predictive of BV. PCR quantification of G. vaginalis and M. hominis from CVL is significantly more sensitive than Amsel criteria for diagnosing BV.

DNA, Bacterial↗
PubMed2002

Routine use of real-time quantitative PCR for laboratory diagnosis of Epstein-Barr virus infections.

Real-time quantitative polymerase chain reaction (PCR) on the LightCycler instrument (LC-PCR) was developed to measure the Epstein-Barr virus (EBV) load in clinical samples. LC-PCR detected two copies of the EBV genome per 500 ng of DNA. Its specificity was confirmed by assays in EBV-negative cell lines, other human herpesviruses and EBV-seronegative individuals. Excellent inter-assay reproducibility of LC-PCR was obtained in 43 samples (r = 0.983). LC-PCR results were compared with a routinely used ELISA-PCR of 150 samples and a good correlation was found (r = 0.956). A total of 88 individuals were studied, including healthy EBV-seropositive adults (n = 32), patients with EBV-associated disease (n = 34), and HIV-infected patients (n = 22); 37.5% of PBMC samples from healthy individuals contained EBV DNA, while no serum sample was positive. The viral load was significantly higher in PBMCS and saliva specimens in patients recently infected with HIV (19 and 39,400 copies/microg DNA, respectively), as well as in AIDS patients (122 and 331,130 copies/microg DNA) than in the control population (0 and 35 copies/microg DNA). This study confirmed that EBV load measurement with LC-PCR is helpful in the management of EBV-related post-transplantation lymphoproliferative disorders and probably of EBV-associated primary central nervous system B-cell lymphoma.

AIDS-Related Opportunistic Infections↗
PubMed2005

Relative transcript quantification by quantitative PCR: roughly right or precisely wrong?

BACKGROUND: When estimating relative transcript abundances by quantitative real-time PCR (Q-PCR) we found that the results can vary dramatically depending on the method chosen for data analysis. RESULTS: Analyses of Q-PCR results from a salmon louse starvation experiment show that, even with apparently good raw data, different analytical approaches 12 may lead to opposing biological conclusions. CONCLUSION: The results emphasise the importance of being cautious when analysing Q-PCR data and indicate that uncritical routine application of an analytical method will eventually result in incorrect conclusions. We do not know the extent of, or have a universal solution to this problem. However, we strongly recommend caution when analysing Q-PCR results e.g. by using two or more analytical approaches to validate conclusions. In our view a common effort should be made to standardise methods for analysis and validation of Q-PCR results.

Animals↗
PubMed2001

Real-time and quantitative PCR: applications to mechanism-based toxicology.

There is increasing awareness that quantitative analysis of changes in molecular targets plays a key role in addressing scientific questions in molecular toxicology, molecular epidemiology, and human risk assessment. One of the emerging technologies that is being used to analyze these molecular targets is real-time and quantitative (RTAQ) polymerase chain reaction (PCR). The aim of this review is to provide the reader with an overview of this technology and to highlight specific applications of this technology to some key areas of molecular toxicology.

Animals↗
PubMed2006

Quantitative PCR on 5 genes reliably identifies CTCL patients with 5% to 99% circulating tumor cells with 90% accuracy.

We previously identified a small number of genes using cDNA arrays that accurately diagnosed patients with Sézary Syndrome (SS), the erythrodermic and leukemic form of cutaneous T-cell lymphoma (CTCL). We now report the development of a quantitative real-time polymerase chain reaction (qRT-PCR) assay that uses expression values for just 5 of those genes: STAT4, GATA-3, PLS3, CD1D, and TRAIL. qRT-PCR data from peripheral blood mononuclear cells (PBMCs) accurately classified 88% of 17 patients with high blood tumor burden and 100% of 12 healthy controls in the training set using Fisher linear discriminant analysis (FLDA). The same 5 genes were then assayed on 56 new samples from 49 SS patients with blood tumor burdens of 5% to 99% and 69 samples from 65 new healthy controls. The average accuracy over 1000 resamplings was 90% using FLDA and 88% using support vector machine (SVM). We also tested the classifier on 14 samples from patients with CTCL with no detectable peripheral involvement and 3 patients with atopic dermatitis with severe erythroderma. The accuracy was 100% in identifying these samples as non-SS patients. These results are the first to demonstrate that gene expression profiling by quantitative PCR on a selected number of critical genes can be employed to molecularly diagnosis SS.

Dermatitis, Atopic↗
PubMed1996

Use of a semi-quantitative PCR for cytomegalovirus DNA as a basis for pre-emptive antiviral therapy in allogeneic bone marrow transplant patients.

The aim of this study was to evaluate the efficacy of pre-emptive antiviral therapy, based on a semi-quantitative nested PCR for cytomegalovirus (CMV) DNA in leukocytes, for the prevention of CMV disease after allogeneic BMT. Fifty-eight patients were prospectively followed with PCR for CMV DNA and antiviral therapy with ganciclovir was initiated after two consecutive positive tests. The levels of CMV DNA were determined by serial dilutions of the positive samples. The probability of detection of CMV DNA was 48.3% and the probability of CMV disease 6% at 100 days after BMT. Patients with CMV disease had higher CMV DNA levels compared with patients without CMV disease (P = 0.001). In comparison to 58 matched historical controls detection of CMV DNA was 5 days earlier (NS) and antiviral therapy could be initiated 10 days earlier in patients followed by PCR (P = 0.05). Pre-emptive antiviral therapy was given to 28 patients in a total of 36 courses. Patients became negative in PCR after 28 of 36 courses (77%). We conclude that PCR for CMV DNA can be used for early detection of CMV infection and as the basis of initiation of pre-emptive antiviral therapy in BMT patients.

Antiviral Agents↗
PubMed2006

Functional analysis of gammaretroviral vector transduction by quantitative PCR.

BACKGROUND: In a clinical setting of gene therapy, quantitative methods are required to determine recombinant viral titres and transgene mRNA expression, avoiding the use of reporter genes. METHODS: We describe procedures based on quantitative polymerase chain reaction (qPCR) designed to assess functional titres of murine leukaemia virus (MLV) vectors, determine proviral copy numbers in transduced cells, and estimate retroviral transgene expression in both target cell lines and mice with transduced chimeric haematopoiesis. RESULTS: Compared to EGFP titration, proviral DNA detection by qPCR was more accurate in assessing the number of infective particles in supernatants, such that average viral titres in terms of proviral copies per cell were two-fold higher. Transgene mRNA expression was directly determined from the vectors used without the need for reporter assays. A new parameter, defined here as the 'transcription index' (TI), served to establish the association between transcribed transgenic mRNA and each proviral insertion. The TI represents the potential expression of every vector or insertion in each cell type, and is thus useful as a control parameter for monitoring preclinical or clinical protocols. CONCLUSIONS: The practical use of qPCR is demonstrated as a valuable alternative to reporter genes for the assessment and surveillance of insertion numbers and transgene expression. In combination with protein expression, this approach should be capable of establishing safer therapeutic gene doses, avoiding the potential side effects of high transduction and expression levels.

3T3 Cells↗
PubMed2003

Determination of HCV genotype by direct sequence analysis of quantitative PCR products.

Hepatitis C virus (HCV) genotyping, combined with quantitative evaluation of HCV RNA, may be beneficial for the management of chronic hepatitis C and in the selection of candidates for interferon treatment. In this study, the COBAS AMPLICOR HCV MONITOR test, a commercially available quantitative assay for HCV RNA, was used. Amplification products obtained from HCV-positive cases were subjected to direct sequencing and genotyping based on seven phylogenetically informative regions within the 5'UTR. Results were compared with those obtained by INNO-LiPA assay. Typing results yielded by both methods were in complete accordance for type and subtype assignment. Twenty-nine of 500 specimens (5.8%) were unclassifiable and belonged to samples with a titer of <70.000 IU, as determined by quantitative assay. Despite this limitation, the overall gain in efficiency, the low rate of test failure and a better resolution of mixed genotypes all constitute a considerable advantage of this system over the commercial hybridization technique for routine clinical laboratory use.

5' Untranslated Regions↗
PubMed2004

Quantitative PCR for DNA identification based on genome-specific interspersed repetitive elements.

We have designed and evaluated a series of class-specific (Aves), order-specific (Rodentia), and species-specific (equine, canine, feline, rat, hamster, guinea pig, and rabbit) polymerase chain reaction (PCR)-based assays for the identification and quantitation of DNA using amplification of genome-specific short and long interspersed elements. Using SYBR Green-based detection, the minimum effective quantitation levels of the assays ranged from 0.1 ng to 0.1 pg of starting DNA template. Background cross-amplification with DNA templates derived from sixteen other species was negligible prior to 30 cycles of PCR. The species-specificity of the PCR amplicons was further demonstrated by the ability of the assays to accurately detect known quantities of species-specific DNA from mixed (complex) sources. The 10 assays reported here will help facilitate the sensitive detection and quantitation of common domestic animal and bird species DNA from complex biomaterials.

Animals↗
PubMed2004

Quantitative PCR analysis reveals a high incidence of large intragenic deletions in the FANCA gene in Spanish Fanconi anemia patients.

Fanconi anaemia is an autosomal recessive disease characterized by chromosome fragility, multiple congenital abnormalities, progressive bone marrow failure and a high predisposition to develop malignancies. Most of the Fanconi anaemia patients belong to complementation group FA-A due to mutations in the FANCA gene. This gene contains 43 exons along a 4.3-kb coding sequence with a very heterogeneous mutational spectrum that makes the mutation screening of FANCA a difficult task. In addition, as the FANCA gene is rich in Alu sequences, it was reported that Alu-mediated recombination led to large intragenic deletions that cannot be detected in heterozygous state by conventional PCR, SSCP analysis, or DNA sequencing. To overcome this problem, a method based on quantitative fluorescent multiplex PCR was proposed to detect intragenic deletions in FANCA involving the most frequently deleted exons (exons 5, 11, 17, 21 and 31). Here we apply the proposed method to detect intragenic deletions in 25 Spanish FA-A patients previously assigned to complementation group FA-A by FANCA cDNA retroviral transduction. A total of eight heterozygous deletions involving from one to more than 26 exons were detected. Thus, one third of the patients carried a large intragenic deletion that would have not been detected by conventional methods. These results are in agreement with previously published data and indicate that large intragenic deletions are one of the most frequent mutations leading to Fanconi anaemia. Consequently, this technology should be applied in future studies on FANCA to improve the mutation detection rate.

Alu Elements↗
PubMed2006

Quantitative PCR assays for mouse enteric flora reveal strain-dependent differences in composition that are influenced by the microenvironment.

The mammalian gastrointestinal (GI) tract is inhabited by over a hundred species of symbiotic bacteria. Differences among individuals in the composition of the GI flora may contribute to variation in in vivo experimental analyses and disease susceptibility. To investigate potential interindividual differences in GI flora composition, we developed real-time quantitative PCR-based assays for the detection of the eight members of the Altered Schaedler Flora (ASF) as representative members of different bacterial niches within the mammalian GI tract. Quantitative and reproducible strain-specific variations in the numbers of the ASF members were observed across 23 different barrier-housed inbred mouse strains, suggesting that the ASF assays can be used as sentinels for changes in GI flora composition. A significant cage effect was also detected. Isogenic mice that cohabited at weaning, whether from the same or different litters, showed little variation in ASF profiles. Conversely, litters split among different cages at weaning showed divergence in ASF profiles after three weeks. Individual ASF profiles, once established, were highly stable over time in the absence of environmental perturbation. Furthermore, cohabitation of different inbred strains maintained most of the interstrain variation in the GI flora, supporting a role of host genetics in determining GI flora composition.

Animals↗
PubMed2001

Evaluation of real-time quantitative PCR for identification and quantification of Chlamydia pneumoniae by comparison with immunohistochemistry.

Chlamydia pneumoniae is a common cause of community-acquired pneumonia and it has been associated with atherosclerosis. C. pneumoniae has usually been diagnosed by serology using a microimmunofluorescence test, but more recently polymerase chain reaction (PCR) has been viewed as an advantageous alternative. We developed a quantitative real-time PCR for detection of C. pneumoniae. Primers were targeted for the pmp4 gene, and the PCR fragment was detected real-time with a fluorescence resonance energy transfer probe set using a LightCycler instrument. The PCR was used on DNA released from 50 microm sections of paraffin-embedded formalin-fixed lung tissue from experimentally infected mice. Thereby, the number of C. pneumoniae genomes was determined. To our knowledge this is the first time quantification of C. pneumoniae DNA has been attempted on paraffin-embedded formalin-fixed tissue. C. pneumoniae-specific immunohistochemistry (IHC) was done on 5 microm sections adjacent to the sections used in PCR, and the number of inclusions were counted in each section. Good correlation was found when comparing results from PCR and IHC, which is in contrast to many previous studies.

Animals↗
PubMed2003

Quantitative PCR analysis of CYP1A induction in Atlantic salmon (Salmo salar).

Environmental pollutants are hypothesized to be one of the causes of recent declines in wild populations of Atlantic salmon (Salmo salar); across Eastern Canada and the United States. Some of these pollutants, such as polychlorinated biphenyls and dioxins, are known to induce expression of the CYP1A subfamily of genes. We applied a highly sensitive technique, quantitative reverse transcription-polymerase chain reaction (RT-PCR), for measuring the levels of CYP1A induction in Atlantic salmon. This assay was used to detect patterns of CYP1A mRNA levels, a direct measure of CYP1A expression, in Atlantic salmon exposed to pollutants under both laboratory and field conditions. Two groups of salmon were acclimated to 11 and 17 degrees C, respectively. Each subject then received an intraperitoneal injection (50 mg kg(-1)) of either beta-naphthoflavone (BNF) in corn oil (10 mg BNF ml(-1) corn oil) or corn oil alone. After 48 h, salmon gill, kidney, liver, and brain were collected for RNA isolation and analysis. All tissues showed induction of CYP1A by BNF. The highest base level of CYP1A expression (2.56 x 10(10) molecules/microg RNA) was found in gill tissue. Kidney had the highest mean induction at five orders of magnitude while gill tissue showed the lowest mean induction at two orders of magnitude. The quantitative RT-PCR was also applied to salmon sampled from two streams in Massachusetts, USA. Salmon liver and gill tissue sampled from Millers River (South Royalston, Worcester County), known to contain polychlorinated biphenyls (PCBs), showed on average a two orders of magnitude induction over those collected from a stream with no known contamination (Fourmile Brook, Northfield, Franklin County). Overall, the data show CYP1A exists and is inducible in Atlantic salmon gill, brain, kidney, and liver tissue. In addition, the results obtained demonstrate that quantitative PCR analysis of CYP1A expression is useful in studying ecotoxicity in populations of Atlantic salmon in the wild.

Animals↗
PubMed1995

Survival and activity of Pseudomonas sp. strain B13(FR1) in a marine microcosm determined by quantitative PCR and an rRNA-targeting probe and its effect on the indigenous bacterioplankton.

Genetically engineered Pseudomonas sp. strain B13(FR1) was released into laboratory-scale marine ecosystem models (microcosms). Survival of the introduced population in the water column and the sediment was determined by plating on a selective medium and by quantitative competitive PCR. The activity of the released bacteria was determined by in situ hybridization of single cells with a specific rRNA-targeting oligonucleotide probe. Two microcosms were inoculated with 10(6) cells ml-1, while an uninoculated microcosm served as a control. The number of Pseudomonas sp. strain B13(FR1) cells decreased rapidly to ca. 10(2) cells ml-1 within 2 days after the release, which is indicative of grazing by protozoa. Three days after the introduction into seawater, cells were unculturable, but PCR continued to detect cells in low numbers. Immediately after the release, the ribosomal content of Pseudomonas sp. strain B13(FR1) corresponded to a generation time of 2 h. The growth rate decreased to less than 0.04 h-1 in 5 days and remained low, probably because of carbon limitation of the cells. Specific amendment of the microcosms with 10 mM 4-chlorobenzoate resulted in a rapid increase of the growth rate and an exponentially increasing number of cells detected by PCR, but not in resuscitation of the cells to a culturable state. The release of Pseudomonas sp. strain B13(FR1) into the microcosms seemed to affect only the indigenous bacterioplankton community transiently. Effects on the community were also apparent from the handling of water during filling of the microcosms and the amendment with 4-chlorobenzoate.

Animals↗
PubMed1999

High-throughput real-time reverse transcription-PCR quantitation of hepatitis C virus RNA.

We describe a rapid and reproducible method for assessment of the hepatitis C virus (HCV) load in serum samples. The method combines Taqman technology (Roche) and the ABI Prism 7700 (Perkin Elmer) real-time sequence detection system. We have optimized a single-tube reverse transcription-PCR (RT-PCR) that contains a dual-labeled fluorogenic probe to quantify the 5' noncoding region (5' NCR) of HCV. The probe contains a fluorescent reporter at the 5' end and a fluorescent quencher at the 3' end. The use of such a probe combined with the 5'-3' nuclease activity of Taq polymerase allows direct quantitation of the PCR product by the detection of a fluorescent reporter released in the course of the exponential phase of the PCR. For accurate quantitation of the number of copies of HCV in samples containing unknown quantities, we have used serial dilutions of a synthetic 5' NCR RNA standard of HCV that was previously quantified with an isotopic tracer. The method has a 5-log dynamic range (10(3) to 10(7)). The coefficient of regression of the standard curve was, on average, 0.98. The intra-assay and the interassay coefficients of variation of the threshold cycle were 1% and 6.2%, respectively. Seventy-nine RNA samples from the sera of infected patients were quantified by this method. Comparison of the results with those obtained by other quantitation methods (the Quantiplex 2.0 branched-DNA assay and the Superquant assay from the National Genetics Institute) revealed a significant correlation with all of the results. The mean values were also statistically comparable. In conclusion, the high sensitivity, simplicity, and reproducibility of the real-time HCV RNA quantitation which allows the screening of large numbers of samples, combined with its wide dynamic range, make this method especially suitable for monitoring of the viral load during therapy and tailoring of treatment schedules.

Adult↗
PubMed2026

Molecular Landscape and Advanced Diagnostic Technologies for BRAF Mutations in Cancer: From Quantitative PCR and ddPCR to CRISPR-Based Platforms.

BRAF mutations are key oncogenic alterations across multiple malignancies, including melanoma, thyroid carcinoma, colorectal cancer, non-small cell lung cancer, glioma, and hairy cell leukemia. The most prevalent variant, BRAF-V600E, induces constitutive activation of the MAPK signaling pathway, promoting tumor progression and influencing therapeutic responsiveness. Accurate detection of BRAF alterations is therefore essential for molecular classification, prognostic assessment, treatment selection, and resistance surveillance. This review summarizes the molecular heterogeneity of BRAF mutations and critically evaluates current diagnostic methodologies. Conventional approaches such as allele-specific PCR and Sanger sequencing are compared with advanced quantitative platforms, including high-resolution melting analysis, droplet digital PCR, and next-generation sequencing, with emphasis on analytical sensitivity, mutation coverage, and clinical applicability. Emerging technologies such as CRISPR-based assays, rolling circle amplification systems, and nanoparticle-based biosensors and point-of-care diagnostic platforms are also discussed for their potential to enhance ultra-sensitive detection, particularly in liquid biopsy settings. These emerging tools are highlighted for their potential to enable ultra-sensitive, rapid, and decentralized mutation detection, particularly in liquid biopsy settings. Key challenges, including intratumoral heterogeneity, low allele-frequency variants, FFPE-associated artifacts, and clonal evolution under therapeutic pressure, are examined within a translational framework. In addition, we examine critical barriers to clinical implementation, including standardization, cost, and global accessibility of molecular diagnostics, and outline potential solutions through scalable technologies and decentralized testing strategies. We propose that optimal BRAF testing requires a mutation subclass-informed and clinically integrated strategy combining comprehensive baseline profiling with longitudinal molecular monitoring. Future diagnostic paradigms will likely integrate multi-omics data and artificial intelligence (AI)-assisted interpretation to refine precision oncology implementation. Looking forward, we propose that optimal BRAF testing will require integration of multi-omics profiling with AI-assisted interpretation, enabling automated variant classification, real-time clinical decision support, and improved prediction of therapeutic response and resistance.

Humans↗
PubMed2006

[Prognosis value of the pp65 antigenemia and semi-quantitative PCR in the detection of the CMV reactivation in bone marrow grafted patients].

In this article a Cytomegalovirus (CMV) antigenemia and semiquantitative PCR retrospective evaluation of 26 bone marrow allo-grafted patients for different haematological disease is reported. Eighteen patients had a CMV reactivation despite a prophylactic treatment, seven of those patients had both positive antigenemia pp65 and positive semi-quantitative CMV PCR. During CMV reactivation, 3 patients developed a CMV disease despite a pre-emptive therapy. The follow up of the antigenemia was performed since D21 until D100 post transplantation, the antigenemia positivity occurred at D53 and was preceded about 7 days by CMV PCR positivity The CMV disease wasn't associated with a high viral load. All patients that had CMV reactivation had a positive CMV serology before the graft, whereas only 37.5% of the patients who did not reactivate had a positive CMV serology. Respectively half patients who reactivated and only 12.5% of those who didn't had a Graft versus host disease (GVHD), witch preceded the reactivation about 21 days in six of the formers. Clinical and biological signs presented by our patients in this cases report, seems to be associated more with the GVHD than with CMV reactivation.

Adolescent↗
PubMed1993

HER-2 and INT-2 amplification estimated by quantitative PCR in paraffin-embedded ovarian cancer tissue samples.

Competitive polymerase chain reaction (PCR) systems were developed for rapid and quantitative estimation of HER-2 (c-erbB-2) and INT-2 oncogene amplification in paraffin-embedded ovarian cancer tissue samples. The beta-globin gene was used as reference and DNA from paraffin-embedded placenta tissue as single copy control. Reliability of the PCR method could be demonstrated by comparing dot blot data with PCR data of identical tumour samples. The PCR method was used to determine HER-2 and INT-2 copy numbers in 196 ovarian cancer samples. HER-2 and INT-2 were found to be amplified in 40 and 19%, respectively. In 8% HER-2 copy numbers were greater than five, but no high INT-2 copies were noted. Kaplan-Meier estimates did not reveal significant association with overall survival. Indirect correlation between HER-2 and INT-2 amplification was observed. The present PCR system is a valuable method for prospective and retrospective studies.

Base Sequence↗