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PubMed2004

Development and application of a real-time quantitative PCR assay for determining CYP1A transcripts in three genera of salmonids.

The expression of CYP1A (cytochrome P4501A) can be induced by a large array of aromatic and organic compounds in teleost fishes. We developed a real-time quantitative PCR assay useful for measuring beta-naphthoflavone (BNF) induction of liver CYP1A mRNA in four salmonid species. First, to obtain necessary information for the design of a cRNA standard, full-length CYP1A cDNA sequences were determined for two Salvelinus species, lake trout (S. namaycush) and brook trout (S. fontinalis). Each cDNA was found to share the same characteristics with known CYP1A sequences of Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss): a start codon, conserved heme-binding region, putative poly-adenylation signal, stop codon, relatively long 3'-untranslated region (UTR; >1 kb), and a protein length of 523 amino acid residues. The brook trout and lake trout CYP1A cDNA's were 2636 and 2672 base pairs (bp) in length and shared greater than 97% coding region sequence identity with Atlantic salmon and rainbow trout CYP1A's. Next, using the generated sequence information, we developed a CYP1A-specific real-time quantitative PCR assay. Primers and a fluorescent-labeled probe were designed from a 68 bp region that was found to be conserved among salmonid CYP1A genes. The assay was designed to allow for simultaneous comparison of CYP1A expression among each experimental group. Finally, groups (n = 4-8) of hatchery-raised Atlantic salmon, brook trout, lake trout, and rainbow trout were given an intraperitoneal injection of a corn oil control, 25 mg kg(-1) BNF, or 50 mg kg(-1) BNF and sacrificed after 48 h. Liver tissue was collected and CYP1A mRNA levels were estimated. In all species, BNF treated fish showed 1.8-3.0 orders of magnitude higher CYP1A than control fish. The CYP1A induction levels were not different in fish treated with both dosages. Mean base levels of CYP1A expression ranged from 7.24 x 10(6) (rainbow trout) to 1.05 x 10(7) (brook trout) transcripts microg(-1) total RNA. Mean induced levels of CYP1A expression ranged from 1.07 x 10(8) (lake trout) to 1.05 x 10(9) (brook trout) trancripts microg(-1) total RNA.

Amino Acid Sequence↗
PubMed2003

Quantitation of human cytomegalovirus in recipients of solid organ transplants by real-time quantitative PCR and pp65 antigenemia.

Human cytomegalovirus (HCMV) infections and anti-HCMV treatment are usually monitored by measuring pp65 antigenemia. This method is time-consuming, labour-intensive and requires skilled operators. We have compared results obtained using real-time Light Cycler quantitative PCR (QPCR) and the pp65 antigen assay on serial samples collected from recipients of solid organ transplants. We collected 198 blood samples from 14 solid organ transplant recipients and assayed them for pp65 antigen and with Light Cycler PCR. HCMV DNA was extracted from leukocytes and measured using primers and probe located in the UL83 region. The quantity of HCMV DNA was calculated using a standard curve prepared from a plasmid containing the target sequence. There was a good correlation between the number of pp65-positive cells and the DNA copy number (r = 0.57, P < 0.0001). A clinical threshold of 50 positive polymorphonuclear leukocytes/200,000 cells was equivalent to two log10 genome copies per capillary by Light Cycler PCR. HCMV DNA was detected before pp65 antigen in three patients at a mean time of 10 days, whereas the two tests were positive simultaneously for eight patients. Both the pp65 antigen data and DNA copy number decreased over time during antiviral treatment, although the QPCR was positive 28.2 days after the pp65 antigen assay had become negative. The real-time Light Cycler quantitative PCR assay is a rapid and labour-saving technique. This molecular method could be useful for monitoring infections and antiviral treatment in recipients of solid organ transplants.

Adult↗
PubMed2003

Strategies for control of trachoma: observational study with quantitative PCR.

BACKGROUND: Antibiotics are an important part of WHO's strategy to eliminate trachoma as a blinding disease by 2020. At present, who needs to be treated is unclear. We aimed to establish the burden of ocular Chlamydia trachomatis in three trachoma-endemic communities in Tanzania and The Gambia with real-time quantitative PCR. METHODS: Conjunctival swabs were obtained at examination from 3146 individuals. Swabs were first tested by the qualitative Amplicor PCR, which is known to be highly sensitive. In positive samples, the number of copies of omp1 (a single-copy C trachomatis gene) was measured by quantitative PCR. FINDINGS: Children had the highest ocular loads of C trachomatis, although the amount of pooling in young age groups was less striking at the site with the lowest trachoma frequency. Individuals with intense inflammatory trachoma had higher loads than did those with other conjunctival signs. At the site with the highest prevalence of trachoma, 48 of 93 (52%) individuals with conjunctival scarring but no sign of active disease were positive for ocular chlamydiae. INTERPRETATION: Children younger than 10 years old, and those with intense inflammatory trachoma, probably represent the major source of ocular C trachomatis infection in endemic communities. Success of antibiotic distribution programmes could depend on these groups receiving effective treatment.

Adolescent↗
PubMed2006

Beepath: an ordered quantitative-PCR array for exploring honey bee immunity and disease.

Honey bees and other insects face many important parasites and pathogens against which they have evolved behavioral, morphological, physiological, and immune-based defenses. To help validate honey bee immune-gene candidates and determine their responsiveness to pathogens, a quantitative-PCR array was developed to measure transcript levels for 48 honey bee and pathogen genes in parallel. It is shown that this array can accurately measure host and pathogen transcript abundance, providing a new tool for assessing the environmental and genetic components behind honey bee immunity. By using common platforms and chemistries (e.g., SYBR-Green intercalating dye), quantitative-PCR arrays such as the one described should have diverse uses in invertebrate pathology.

Animals↗
PubMed2001

[Real-Time Quantitative PCR and Detection of Minimal Residual Disease in Blood Malignancy]

Real-time quantitative PCR (RQ-PCR) is a recently developed technique for nuclear acid quantification, in which conventional PCR and probe hybridization are integrated together. A specific fluorescent-labeled probe, binded with DNA/cDNA target, is supplemented into the reaction system of PCR. During the extension phase of PCR amplification, the specific fluorescent-labeled probe is cleaved by the 5'-->3' exonuclease activity of Taq polymerase, producing a fluorescent signal that can be detected. The amount of fluorescence released in the reaction is proportional to the target sequence number. Thus, an exactly and absolutely quantitative assessment of initial target DNA/cDNA copy number can be made in a closed tube assay system. This technique eliminates the need for post-PCR sample processing which is necessary in conventional PCR and decreases the probability of contamination which is inevitable in conventional PCR. Moreover, it reduces the potiental for false positive or negative results by utilizing the hybridization of a probe to sequences internal to amplification primers. RQ-PCR is proved to be a sensitive, specific, reliable and convenient tool for the quantification of minimal residual diseases (MRD) of hematological malignancies by recent studies. This article reviews the principle of RQ-PCR and its application in quantitative detection of MRD of hematological malignancies such as acute myelogenous leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myelogenous leukemia (CML) and malignant lymphoma.

Journal Article↗
PubMed2002

Using real-time, quantitative PCR for rapid genotyping of the steroid 21-hydroxylase gene in a north Florida population.

The most common cause of congenital adrenal hyperplasia is steroid 21-hydroxylase deficiency. The molecular genetics of this disease are such that genotyping is a potentially useful tool in its diagnosis. An assay was developed using real-time, quantitative PCR to detect deletions of the steroid 21-hydroxylase gene (CYP21A2). This assay was able to detect heterozygous gene deletions with an alpha error rate of less than 5%, with a power greater than 95%. When combined with allele-specific PCR, genotyping for the nine most common mutations can be completed within hours of blood sampling. This technique was used to study subjects with 21-hydroxylase deficiency in North Florida. Twenty-eight subjects with congenital adrenal hyperplasia, seven first-degree relatives and thirteen normal subjects, were characterized. Of 96 chromosomes, 69 abnormal alleles were identified. Among unrelated abnormal alleles, the frequency of specific mutations was 28% for a gene deletion, 24% for the intron 2 splice mutation, 10% for ile172asn, 8% each for val281leu and the exon 6 cluster, and 6% for gln318x mutations. These frequencies, as well as the genotype/phenotype correlation, were similar to those found in comparable populations. The utility of genotyping in the diagnosis of 21-hydroxylase deficiency is increased by the rapidity of the analysis. With quantitative PCR, the need for more expensive and time consuming Southern blot analysis is reduced and limited to the clarification of certain genotypes. Faster results will allow for more timely initiation of appropriate therapy and limit the exposure of potentially unnecessary therapy.

Blood Preservation↗
PubMed2005

Generally applicable methods to purify intracellular coccidia from cell cultures and to quantify purification efficacy using quantitative PCR.

The objective of this study was to evaluate the utility of a simple, efficient, and rapid method for the isolation of Sarcocystis neurona merozoites and Besnoitia darlingi tachyzoites from cultured cells. The efficacy of this purification method was assessed by microscopy, SDS-PAGE, Western blotting, immuno-fluorescence, and three novel quantitative PCR assays. Culture medium containing host cell debris and parasites was eluted through PD-10 desalting columns. This purification method was compared to alternatives employing filtration through a cellulose filter pad or filter paper. The estimated recovery of S. neurona merozoites purified by the column method was 82% (+/-3.7) of the original merozoites with 97.5% purity. In contrast, estimated recovery of S. neurona merozoites purified by filter pad and filter paper was 40% and 30% with 76% and 83% purity, respectively. The same procedures were applied to purify B. darlingi tachyzoites from cultured cells. Of the original cultured B. darlingi tachyzoites, 94% (+/-2.5) were recovered from the PD-10 column with 96.5%, purity whereas percentage recovery of B. darlingi tachyzoites purified by filter pad and filter paper were 51% and 35% with 84% and 88% purity, respectively. All described methods maintained sterility so that purified parasites could be subsequently cultured in vitro. However, purification using a PD-10 column minimized parasite loss and the loss of viability as determined by the trypan blue dye exclusion assay, the rate of parasite production, and plaque forming efficiency in cell culture. Moreover, column-purified parasites improved the sensitivity of an immuno-fluorescent (IFA) analysis and real-time quantitative PCR assays targeted to parasite 18S ribosomal DNA and hsp70 genes. This technique appears generally applicable for purifying coccidia grown in cell cultures.

Animals↗
PubMed2004

Analysis of a quantitative PCR assay for CMV infection in liver transplant recipients: an intent to find the optimal cut-off value.

BACKGROUND: Preemptive therapy required highly predictive tests for CMV disease. CMV antigenemia assay (pp65 Ag) has been commonly used for rapid diagnosis of CMV infection. Amplification methods for early detection of CMV DNA are under analysis. OBJECTIVES: To compare two diagnostic methods for CMV infection and disease in this population: quantitative PCR (qPCR) performed in two different samples, plasma and leukocytes (PMNs) and using a commercial diagnostic test (COBAS Amplicor Monitor Test) versus pp65 Ag. STUDY DESIGN: Prospective study conducted in liver transplant recipients from February 2000 to February 2001. RESULTS: Analyses were performed on 164 samples collected weekly during early post-transplant period from 33 patients. Agreements higher than 78% were observed between the three assays. Optimal qPCR cut-off values were calculated using ROC curves for two specific antigenemia values. For antigenemia >or=10 positive cells, the optimal cut-off value for qPCR in plasma was 1330 copies/ml, with a sensitivity (S) of 58% and a specificity (E) of 98% and the optimal cut-off value for qPCR-cells was 713 copies/5x10(6) cells (S:91.7% and E:86%). Using a threshold of antigenemia >or=20 positive cells, the optimal cut-off values were 1330 copies/ml for qPCR-plasma (S 87%; E 98%) and 4755 copies/5x10(6) cells for qPCR-cells (S 87.5%; E 98%). Prediction values for the three assays were calculated in patients with CMV disease (9 pts; 27%). Considering the assays in a qualitative way, the most sensitive was CMV PCR in cells (S: 100%, E: 54%, PPV: 40%; NPV: 100%). Using specific cut-off values for disease detection the sensitivity, specificity, PPV and NPV for antigenemia >or=10 positive cells were: 89%; 83%; 67%; 95%, respectively. For qPCR-cells >or=713 copies/5x10(6) cells: 100%; 54%; 33% and 100% and for plasma-qPCR>or=1330 copies/ml: 78%, 77%, 47%, 89% respectively. CONCLUSIONS: Optimal cut-off for viral load performed in plasma and cells can be obtained for the breakpoint antigenemia value recommended for initiating preemptive therapy with high specificities and sensitivities. Diagnostic assays like CMV pp65 Ag and quantitative PCR for CMV have similar efficiency and could be recommended as methods of choice for diagnosis and monitoring of active CMV infection after transplantation.

Antigens, Viral↗
PubMed2002

Multiplex quantitative PCR using self-quenched primers labeled with a single fluorophore.

Multiplex quantitative PCR based on novel design of fluorescent primers is described. Fluorogenic primers are labeled with a single fluorophore on a base close to the 3' end with no quencher required. A tail of 5-7 nt is added to the 5' end of the primer to form a blunt-end hairpin when the primer is not incorporated into a PCR product. This design provides a low initial fluorescence of the primers that increases up to 8-fold upon formation of the PCR product. The hairpin oligonucleotides (DeltaG from -1.6 to -5.8 kcal/mol) may be as efficient as linear primers and provide additional specificity to the PCR by preventing primer-dimers and mispriming. Multiple fluorogenic primers were designed by specialized software and used for real-time quantitation of c-myc and IL-4 cDNAs in the presence of reference genes such as beta-actin, GAPDH and 18S rRNA. Targets of 10-10(7) copies were detected with precision in PCR using FAM-labeled primers for variable genes and JOE-labeled primers for the reference genes. This method was also used to detect single nucleotide polymorphism of the human retinal degeneration gene by allele-specific PCR with end-point detection using a fluorescent plate reader or a UV-transilluminator. We conclude that fluorogenic mono-labeled primers are an efficient and cost-effective alternative to FRET-labeled oligonucleotides.

Alleles↗
PubMed2005

Qualitative and quantitative PCR methods for event-specific detection of genetically modified cotton Mon1445 and Mon531.

Based on the DNA sequences of the junctions between recombinant and cotton genomic DNA of the two genetically modified (GM) cotton varieties, herbicide-tolerance Mon1445 and insect-resistant Mon531, event-specific primers and probes for qualitative and quantitative PCR detection for both GM cotton varieties were designed, and corresponding detection methods were developed. In qualitative PCR detection, the simplex and multiplex PCR detection systems were established and employed to identify Mon1445 and Mon531 from other GM cottons and crops. The limits of detection (LODs) of the simplex PCR were 0.05% for both Mon1445 and Mon531 using 100 ng DNA templates in one reaction, and the LOD of multiplex PCR analysis was 0.1%. For further quantitative detection using TaqMan real-time PCR systems for Mon1445 and Mon531, one plasmid pMD-ECS, used as reference molecule was constructed, which contained the quantitative amplified fragments of Mon1445, Mon531, and cotton endogenous reference gene. The limits of quantification (LOQs) of Mon1445 and Mon531 event-specific PCR systems using plasmid pMD-ECS as reference molecule were 10 copies, and the quantification range was from 0.03 to 100% in 100 ng of the DNA template for one reaction. Thereafter, five mixed cotton samples containing 0, 0.5, 0.9, 3 and 5% Mon1445 or Mon531 were quantified using established real-time PCR systems to evaluate the accuracy and precision of the developed real-time PCR detection systems. The accuracy expressed as bias varied from 1.33 to 8.89% for tested Mon1445 cotton samples, and from 2.67 to 6.80% for Mon531. The precision expressed as relative standard deviations (RSD) were different from 1.13 to 30.00% for Mon1445 cotton, and from 1.27 to 24.68% for Mon531. The range of RSD was similar to other laboratory results (25%). Concluded from above results, we believed that the established event-specific qualitative and quantitative PCR systems for Mon1445 and Mon531 in this study are acceptable and suitable for GM cotton identification and quantification.

Animals↗
PubMed2000

Kinetics of early therapeutic response as measured by quantitative PCR predicts survival in a murine xenograft model of human T cell acute lymphoblastic leukemia.

The identification of prognostic parameters and surrogate markers for defining patient risk has been beneficial in effectively guiding therapy and increasing the survival of leukemia patients. It has been hypothesized that the therapeutic response, as measured by a change in tumor burden during therapy, might serve as a new surrogate marker of survival. Here we describe the development of a murine SCID xenograft model of human T cell acute lymphoblastic leukemia (T-ALL), and the use of a sensitive, quantitative PCR assay for the measurement of tumor levels to investigate the relationships between tumor burden quantification, therapeutic response and survival. Animals engrafted with the CCRF-CEM (CEM) human T-ALL cell line develop leukemia that closely resembles the human disease. Quantitative PCR detects the expanding tumor mass in the peripheral blood of the animals several weeks before death. In response to induction therapy with chemotherapeutic agents, both the level of minimal residual disease (MRD) in peripheral blood at the end of therapy and the rate of tumor reduction in peripheral blood during therapy strongly correlated with animal survival. Thus, these surrogate markers, which can be measured during the early stages of therapy, may help improve patient survival through dynamic risk stratification.

Animals↗
PubMed2003

Identification of three novel OA1 gene mutations identified in three families misdiagnosed with congenital nystagmus and carrier status determination by real-time quantitative PCR assay.

BACKGROUND: X-linked ocular albinism type 1 (OA1) is caused by mutations in OA1 gene, which encodes a membrane glycoprotein localised to melanosomes. OA1 mainly affects pigment production in the eye, resulting in optic changes associated with albinism including hypopigmentation of the retina, nystagmus, strabismus, foveal hypoplasia, abnormal crossing of the optic fibers and reduced visual acuity. Affected Caucasian males usually appear to have normal skin and hair pigment. RESULTS: We identified three previously undescribed mutations consisting of two intragenic deletions (one encompassing exon 6, the other encompassing exons 7-8), and a point mutation (310delG) in exon 2. We report the development of a new method for diagnosis of heterozygous deletions in OA1 gene based on measurement of gene copy number using real-time quantitative PCR from genomic DNA. CONCLUSION: The identification of OA1 mutations in families earlier reported as families with hereditary nystagmus indicate that ocular albinism type 1 is probably underdiagnosed. Our method of real-time quantitative PCR of OA1 exons with DMD exon as external standard performed on the LightCycler trade mark allows quick and accurate carrier-status assessment for at-risk females.

Adult↗
PubMed2001

Determining liver stage parasite burden by real time quantitative PCR as a method for evaluating pre-erythrocytic malaria vaccine efficacy.

The detection and quantitation of blood stage parasitaemia is typically used as a surrogate endpoint for estimating the efficacy of vaccines targeted against the hepatic stage, as well as the erythrocytic stage, of the parasite. However, this does not provide an adequate means of evaluating the efficacy of vaccines, which may be only partially effective at the liver-stage. This is a particular concern for effective evaluation of immune enhancement strategies for candidate pre-erythrocytic stage vaccines. Here, we have developed and validated a method for detecting and quantitating liver stage parasites, using the TaqMan fluorescent real-time quantitative PCR system (PE Applied Biosystems). This method uses TaqMan primers designed to the Plasmodium yoelii 18S rRNA gene and rodent GAPDH to amplify products from infected mouse liver cDNA. The technique is highly reproducible as demonstrated with plasmid controls and capable of efficiently quantitating liver-stage parasite burden following a range of sporozoite challenge doses in strains of mice, which differ in their susceptibility to sporozoite infection. We have further demonstrated the capacity of this technique to evaluate the efficacy of a range of pre-erythrocytic stage vaccines. Our data establish this quantitative real-time PCR assay to be a fast and reproducible way of accurately assessing liver stage parasite burden and vaccine efficacy in rodent malaria models.

Animals↗
PubMed2005

Quantitative PCR for detection of the OT-1 transgene.

BACKGROUND: Transgenic TCR mice are often used experimentally as a source of T cells of a defined specificity. One of the most widely used transgenic TCR models is the OT-1 transgenic mouse in which the CD8+ T cells express a TCR specific for the SIINFEKL peptide of ovalbumin presented on kb. Although OT-1 CD8+ can be used in a variety of different experimental settings, we principally employ adoptive transfer and peptide-driven expansion of OT-1 cells in order to explore the distribution and fate of these antigen-specific OT-1 T cells. We set out to develop a quantitative PCR assay for OT-1 cells in order to assess the distribution of OT-1 CD8+ T cells in tissues that are either intrinsically difficult to dissociate for flow cytometric analysis or rendered incompatible with flow cytometric analysis through freezing or fixation. RESULTS: We show excellent correlation between flow cytometric assessment of OT-1 cells and OT-1 signal by qPCR assays in cell dilutions as well as in in vivo adoptive transfer experiments. We also demonstrate that qPCR can be performed from archival formalin-fixed paraffin-embedded tissue sections. In addition, the non-quantitative PCR using the OT-1-specific primers without the real-time probe is a valuable tool for OT-1 genotyping, obviating the need for peripheral blood collection and subsequent flow cytometric analysis. CONCLUSION: An OT-1 specific qPCR assay has been developed to quantify adoptively transferred OT-1 cells. OT-1 qPCR to determine cell signal is a valuable adjunct to the standard flow cytometric analysis of OT-1 cell number, particularly in experimental settings where tissue disaggregation is not desirable or in tissues which are not readily disassociated.

Adoptive Transfer↗
PubMed2002

Comparison of cell culture systems for duck hepatitis B virus using SyBr green quantitative PCR.

The Hepadnaviridae family contains DNA viruses such as human hepatitis B virus (HBV), woodchuck hepatitis B virus (WHV), and duck hepatitis B virus (DHBV). DHBV is distributed in both wild and domestic ducks. HBV is a worldwide health problem with carriers at risk of developing cirrhosis and liver cancer. All medical staff and scientists working with HBV must be vaccinated, because of its highly contagious nature. DHBV is a safe surrogate for HBV because of their similarities. Several cell culture systems have been developed to study anti-DHBV drugs and disinfectants. However, differences in their capabilities to support DHBV propagation have not been reported. Therefore, a sensitive and reproducible quantitative PCR based on SyBr green dye was developed. This system does not need electrophoresis for analysis of PCR products, thus reducing processing time and potential for cross-contamination. It allowed precise quantification of DHBV over 8-logarithm dynamic range with a good correlation (R(2) = 0.9689) and showed minimal run-to-run deviation. Sensitivity was 820 copies of DHBV genome and specificity was confirmed by melting curve analysis. It demonstrated good repeatability in quantification of DHBV loads from serum of infected ducks. This assay compared DHBV yields from different cultured cells. All cells had similar kinetic curves for DHBV replication and replication peaks appeared 4 days post-infection. Duck embryonic hepatocytes showed the highest (P > 0.05) replication peak for DHBV. Therefore, duck embryonic hepatocytes and quantitative PCR based on SyBr green dye are a good choice for anti-DHBV drug and disinfectant testing.

Animals↗
PubMed2004

Analysis of CBP (CREBBP) gene deletions in Rubinstein-Taybi syndrome patients using real-time quantitative PCR.

Rubinstein-Taybi syndrome (RTS) is a well-defined syndrome characterized by facial abnormalities, broad thumbs, broad big toes, and growth and mental retardation as the main clinical features. RTS was shown to be associated with disruption of the CREB-binding protein gene CBP (CREBBP), either by gross chromosomal rearrangements or by point mutations. Translocations and inversions involving chromosome band 16p13.3 form the minority of CBP mutations, whereas microdeletions occur more frequently (about 10%). Most deletion studies in RTS are performed by FISH analysis, and five cosmids must be used to cover the whole of the CBP gene, which spreads over 150 kb. Here we report the design of gene dosage assays by real-time quantitative PCR that are targeted on three exons located respectively at the 5' end (exon 2), in the middle (exon 12), and at the 3' end (exon 30) of the CBP gene. This technique proved to be efficient and powerful in finding deletions and complementary to the other available techniques, since it allowed us to identify deletions at the 3' end of the gene that had been missed by FISH analysis, and to refine some deletion breakpoints. Our results therefore suggest that real-time quantitative PCR is a useful technique to be included in the deletion search in RTS patients.

CREB-Binding Protein↗
PubMed2004

Real-time quantitative PCR as a routine method for screening large rearrangements in Rett syndrome: Report of one case of MECP2 deletion and one case of MECP2 duplication.

Mutations in the X-linked methyl-CpG-binding protein 2 gene (MECP2) are found in 70-80% of cases of classical Rett syndrome (RTT) and in about 50% of cases of preserved speech variant (PSV). This high percentage of MECP2 mutations, especially in classical RTT cases, suggests that another major RTT locus is unlikely. Missed mutations may be due to the limited sensitivity of the methodology used for mutation scanning and/or the presence of intronic mutations. In a double-copy gene, such as MECP2 in females, current methodologies (e.g., DGGE, SSCP, DHPLC, direct sequencing) are prone to miss gross rearrangements. Three previous reports during 2001-2003 have shown the presence of large deletions in a fraction of MECP2-negative classical RTT patients. We developed a reliable, single tube, quantitative PCR assay for rapid determination of MECP2 gene dosage. This method involves a multiplex reaction using a FAM labeled TaqMan probe with a TAMRA quencher derived from MECP2 exon 4 and two primers derived from the same exon and RNAaseP as an internal reference. The copy number of the MECP2 gene was determined by the comparative threshold cycle method (ddCt). Each sample was run in quadruplicate. We validated this assay through the analysis of 30 healthy controls (15 female and 15 male) and we then applied this method to eight classical RTT and six PSV patients, all negative for MECP2 mutations. We identified gross rearrangements in two patients: a deletion in a classical RTT patient and a duplication in a PSV patient. Our results confirm that a fraction of MECP2-negative RTT cases have MECP2 gross rearrangements and we propose real-time quantitative PCR as a simple and reliable method for routine screening of MECP2 in addition to DHPLC analysis.

Adult↗
PubMed2006

Influence of segmenting fluids on efficiency, crossing point and fluorescence level in real time quantitative PCR.

The two-phase segmented flow approach to the processing and quantitative analysis of biological samples in microdevices offers significant advantages over the single-phase continuous flow methodology. Despite this, little is known about the compatibility of samples and reactants with segmenting fluids, although a number of investigators have reported reduced yield and inhibition of enzymatic reactions depending on the segmenting fluid employed. The current study addresses the compatibility of various segmenting fluids with real time quantitative PCR to understand the physicochemical requirements of this important reaction in biotechnology. The results demonstrate that creating a static segmenting fluid/PCR mix interface has a negligible impact on the reaction efficiency, crossing threshold and end fluorescence levels using a variety of segmenting fluids. The implication is then that the previously reported inhibitory effects are the result of the dynamic motion between the segmenting fluid and the sample in continuously flowing systems. The results presented here are a first step towards understanding the limitations of the segmented flow methodology, which are necessary to bring this approach into mainstream use.

Complex Mixtures↗