Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Quantitative PCR”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,603 records · Page 89Linked to original sources
PubMed1994

Mixed cryoglobulinemia and hepatitis C virus.

BACKGROUND: Mixed cryoglobulinemia (MC) is frequently associated with clinical and biological evidence of liver disease and has recently been reported in cases of hepatitis C virus (HCV) infection. The aim of this study was to assess prospectively in a large series of MC patients: (1) the prevalence of HCV markers (anti-HCV antibodies and HCV RNA in serum and cryoprecipitate); (2) the main clinical, biologic and liver histologic features in patients with or without HCV infection. PATIENTS: One hundred fifteen consecutive unselected MC patients were studied: 45% had well-defined underlying diseases ("nonessential" MC). Fifty-five percent with no cause of MC were considered to have "essential" MC and were subjected to in-depth examination. METHODS: Patients were considered to have MC if two successive determinations of their serum cryoglobulin level were above 0.05 g/L. Anti-HCV antibodies (Ab) were detected in all patients by second-generation tests (ELISA, RIBA). We also looked for HCV RNA sequences amplified by polymerase chain reaction (PCR) in the sera and cryoprecipitates of 39 patients; HBs antigen, anti-HBs Ab and anti-HBc Ab in all patients; and HBV DNA in 20 sera and 17 cryoprecipitates. Quantitative HCV Ab and RNA studies were performed on whole serum, cryoprecipitates, and supernatants. Clinical features were recorded retrospectively for each patient. Liver biopsies from 23 anti-HCV Ab-positive and 7 anti-HCV Ab-negative patients were examined histologically, with qualitative and quantitative analysis. RESULTS: Anti-HCV Ab were found in 47/115 (41%) patients by ELISA and RIBA: 33/63 (52%) essential MC and 14/52 (27%) nonessential MC. Among the 63 essential MC patients, the 33 anti-HCV Ab-positive (Group 1) were compared to the 30 anti-HCV Ab-negative patients (Group 2). Group 1 patients had more cutaneous involvement (Raynaud's phenomenon, purpura, livedo, distal ulcers, or gangrenous changes) (17 versus 5: p = 0.004), higher alanine aminotransferase levels (110 +/- 22 versus 41 +/- 10 IU; p < 0.005), higher serum cryoglobulin levels (0.35 +/- 0.07 versus 0.12 +/- 0.04 g/L; p = 0.01), lower CH50 (28 +/- 3 versus 44 +/- 2 CH50/mL; p = 0.0001) and lower C4 levels (0.20 +/- 0.02 versus 0.29 +/- 0.03 g/L; p < 0.04). The prevalence of HBV serum markers was low in both groups, and HBV DNA was never detected in any of the sera and cryoprecipitates tested. HCV RNA sequences were detected in 10/16 (63%) sera and 12/16 (75%) cryoprecipitates from Group 1 patients, whereas they were not in the sera or cryoprecipitates from 23 Group 2 patients. Using quantitative PCR, HCV RNA in cryoprecipitates was concentrated 20 to 100 times despite the absence of significant anti-HCV Ab concentration in these samples. Histologic examination of liver biopsies revealed a spectrum of lesions ranging from chronic active hepatitis to cirrhosis, but Knodell's score did not differ between the groups. CONCLUSION: (1) About 50% of the essential MC patients had anti-HCV Ab, and these patients had more severe cryoglobulinemia-associated clinical and biological signs; (2) HCV RNA sequences were found in the large majority of sera and cryoprecipitates from patients with essential MC and anti-HCV Ab and were more concentrated in cryoprecipitates than in supernatants. These results suggest a role for HCV in the pathogenesis of MC and indicate that many cases of essential MC may be secondary to HCV infection and thus nonessential.

Adult↗
PubMed2004

Molecular and functional analysis of hyperpolarisation-activated nucleotide-gated (HCN) channels in the enteric nervous system.

Hyperpolarisation-activated non-specific cation currents (Ih currents) are important for the regulation of cell excitability. These currents are carried by channels of the hyperpolarisation-activated nucleotide-gated (HCN) family, of which there are four known subtypes. In the enteric nervous system (ENS), the Ih current is prominent in AH neurons. We investigated the expression and localization of HCN isoforms in the ENS of mice, rats and guinea-pigs. HCN1, HCN2 and HCN4 were expressed in enteric neurons. Immunoreactivity for HCN1 was observed on neuronal cell membranes of Dogiel type II neurons in rat and mouse. HCN2 channel immunoreactivity occurred in the majority of enteric neurons in the guinea-pig, rat and mouse. Immunoreactivity for HCN4 protein was revealed on the cell membranes of many neurons, including Dogiel type II neurons, in the guinea-pig. HCN4 was expressed by glial cells in guinea-pig. There was no evidence of HCN3 channel protein in any species with either immunohistochemistry or Western analysis. RT-PCR (polymerase chain reaction) using mouse HCN primers revealed mRNA for all four channels in the longitudinal muscle plus myenteric plexus of mouse distal colon. Sequencing confirmed the identity of the mRNA. Quantitative PCR demonstrated that HCN2 was the most highly expressed HCN channel subtype in the myenteric plexus of mouse distal colon. HCN1 and HCN4 were expressed at lower levels. HCN3 subtype mRNA was 0.2% of HCN2. We used intracellular recording to identify neurons having Ih currents and intracellular dye filling to locate the neurons for the immunohistochemical determination of channel expression. AH neurons with Ih currents were HCN2 and HCN4 channel positive. There was no correlation between the magnitude of the Ih and intensity of channel immunoreactivity. Our results indicate that HCN1, 2 and 4 genes and protein are expressed in the ENS. AH/Dogiel type II neurons, which have a prominent Ih, express HCN2 and 4 in guinea-pig and HCN1 and 2 in mouse and rat.

Animals↗
PubMed1998

Lipopolysaccharide-induced expression of IP-10 mRNA in rat brain and in cultured rat astrocytes and microglia.

Using mRNA differential display technique, we have found a differentially expressed band in rat brain, designated HAP2G1, which was the strongest one induced in response to peripheral administration of lipopolysaccharide (LPS). Sequence analysis showed that HAP2G1 cDNA is the rat homologue of the human alpha-chemokine IP-10. Using RT-PCR technique and in situ hybridization, we demonstrate that IP-10 mRNA was expressed only in brain tissue of rats treated with LPS and not in control brain tissue. Using semi-quantitative PCR, we found that both cultured astrocytes and microglia express IP-10 mRNA after treatment with LPS. LPS-induced IP-10 mRNA reached peak levels in rat brain and in cultured microglia at approximately 3 h after treatment with LPS. At 10 h, IP-10 mRNA was markedly decreased, and at 24 h it was low but still detectable by PCR or in situ hybridization. In contrast to unstimulated microglia, unstimulated astrocytes constitutively expressed IP-10 mRNA at a low level. Increased IP-10 expression could possibly be involved in the microglia response to inflammatory stimuli in vivo.

Animals↗
PubMed1994

HIV-1 reverse transcription in cord blood lymphocytes: implications for infection of newborns.

We previously demonstrated that progeny virions are not produced after infection of adult quiescent peripheral blood lymphocytes (PBLs) by human immunodeficiency virus type 1 (HIV-1). Molecular analysis revealed that the nonproductive nature of this infection is due to failure to complete reverse transcription of the viral genome. In this study, we examined HIV-1 reverse transcription in quiescent lymphocytes from umbilical cord blood (CBLs). Using the polymerase chain reaction (PCR) to detect the presence of reverse transcription intermediates, we found that as in PBLs from adults, reverse transcription is not completed in quiescent CBLs; instead, a partial reverse transcript is formed. Quantitative PCR analysis also showed that fewer partial reverse transcripts were found in CBLs than in PBLs. Although the relevance of this restriction in reverse transcription to vertical transmission is unclear, these data suggest that the rapid progression of disease in infected children is not due to increased permissiveness of the lymphocytes of newborns for HIV-1 infection.

Adult↗
PubMed2006

Improved DOP-PCR-based representational whole-genome amplification using quantitative real-time PCR.

In many cases, only a minute amount of partially degraded genomic DNA can be extracted from archived clinical samples. Diverse whole-genome amplification methods are applied to provide sufficient amount of DNA for comparative genome hybridization, single-nucleotide polymorphism, and microsatellite analyses. In these applications, the reliability of the amplification techniques is particularly important. In PCR-based approaches, the plateau effect can seriously alter the original relative copy number of certain chromosomal regions. To eliminate this distorting effect, we improved the standard degenerate oligonucleotide-primed PCR (DOP-PCR) technique by following the amplification status with quantitative real-time PCR (QRT-PCR). With real-time detection of the products, we could eliminate DNA overamplification. Probes were prepared from 10 different tumor samples: primary and metastatic melanoma tissues, epidermoid and bronchioloalveolar lung carcinomas, 2 renal cell carcinomas, 2 colorectal carcinomas, and a Conn and Cushing adenoma. Probes were generated by using nonamplified and amplified genomic DNA with DOP-PCR and DOP-PCR combined with QRT-PCR. To demonstrate the reliability of the QRT-PCR based amplification protocol, altogether 152 relative copy number changes of 44 regions were determined. There was 85.6% concordance in copy number alterations between the QRT-PCR protocol and the nonamplified samples, whereas this value was only 63.8% for the traditional DOP-PCR. Our results demonstrate that our protocol preserves the original copy number of different chromosomal regions in amplified genomic DNA than standard DOP-PCR techniques more accurately.

DNA Primers↗
PubMed2006

[Expression of selected genes involved in transport of ions in papillary thyroid carcinoma].

INTRODUCTION: The studies of papillary thyroid cancer (PTC) gene expression profile have shown changes in expression of genes involved in transport of several ions. The aim of our study was a real-time PCR evaluation of three of them: KCNJ2, SLC4A4 and SLC34A2. MATERIAL AND METHODS: The analysis was carried out in PTC tumors and normal thyroid samples gained from 38 patients. Real-time quantitative PCR (Q-PCR) was performed (Taqman) with beta-glucuronidase (GUS) as the reference gene. RESULTS: We observed 20 x increase of SLC34A2 expression in PTC. This gene encodes Na+/PO(4) (3-) cotransporter. Considerable increase of gene expression has been shown also for KCNJ2, encoding a potassium ion channel. SLC4A4, which encodes the Na+/HCO(3) (2-) cotransporter, exhibited a 7-fold decrease in PTC. CONCLUSIONS: The performed study revealed that SLC34A2 gene exhibited the most distinct change in expression and may become a molecular marker of papillary thyroid cancer.

Adolescent↗
PubMed1991

[Applications of PCR in molecular immunology].

Since a wide panel of monoclonal antibodies specific for antigen receptor variable (V) regions is unavailable, PCR has emerged as the primary analytical method for determining the expressed repertoire of T- and B-lymphocyte populations. Furthermore, PCR amplification of immunoglobulin V-region genes is exploited for the production of monoclonal antibodies by repertoire cloning as a replacement of the B-cell-hybridoma technique and for the humanization of murine monoclonal antibodies. The heavy and light chains of immunoglobulins as well as the transmembrane-protein chains which build the heterodimeric T-cell receptors consist of a constant and of an antigen-binding variable segment. Conventional PCR makes use of two specific oligonucleotide primers that flank a sequence of interest to prime replication of the bracketed DNA. In the case of antigen receptor encoding DNA one flanking sequence of the variable region is unknown or heterogeneous. Therefore, modifications of the conventional PCR had to be developed for the cloning and analysis of antigen-receptor DNA: (1) the usage of known V-gene segments by a lymphocyte population can be analyzed by quantitative PCR with panels of V-specific primers; (2) since PCR primers do not have to be completely complementary to the target DNA, it is possible to amplify a variety of different V regions with degenerate primers for relatively conserved sequences of the V genes; (3) enzymatic addition of a homopolymeric dG tail to the variable end of the antigen receptor DNA makes the amplification completely independent of the structure of the variable region because the dG-tailed DNA can be amplified with a nonspecific poly-dC oligonucleotide combined with a specific primer for the constant gene segment of interest.

Allergy and Immunology↗
PubMed1995

Analysis of human 65 kD heat shock protein mRNA using polymerase chain reaction in synovia of rheumatoid arthritis patients.

To examine the role of 65kD heat shock protein (HSP) on the etiology of RA, we investigated the expression of 65kD HSP mRNA in the synovia of patients with rheumatoid arthritis (RA). Synovial membranes from 10 patients with RA and 2 patients with non-inflammatory arthritis were examined for the expression of the 65kD HSP mRNA by the reverse transcription-polymerase chain reaction (RT-PCR) method. The mRNA of 65kD HSP was detected in all 10 samples from the RA patients and in the 2 samples from the patients with non-RA using semi-quantitative PCR and the difference in its expression level between the two groups was found not to be significant. By sequence analysis of the 412 bp PCR products, the 65kD HSP protein was confirmed to be human-origin. These data suggest that the 65kD HSP expressed in rheumatoid synovia is human origin and the lymphocytic reaction to the HSP may play an important role in the pathogenesis of RA.

Adult↗
PubMed1998

Frequent aberration of FHIT gene expression in acute leukemias.

We analyzed the mRNA expression of the FHIT gene by reverse transcription-PCR (RT-PCR) in 54 cases of acute lymphoblastic leukemia (ALL; 11 cases of T-cell ALL [T-ALL] and 43 cases of non-T-ALL) and 40 cases of acute myeloid leukemia (AML). In 46% of the ALL cases and 55% of the AML cases, FHIT expression was absent or markedly decreased. Only abnormal short bands were detected in 30% of the ALL cases and 5% of the AML cases. Eighteen of 19 abnormal transcripts had the same fusion of exons 2-7, and all lacked the starting codon in exon 5. No obvious normal-sized PCR products were detected in cases exhibiting abnormal transcripts. These findings suggest that the expression of functional FHIT protein was lost in the majority of ALL (76%) and AML (60%) cases. Differential quantitative PCR of exons 3-9 of the FHIT gene and RT-PCR of the PTPRG gene, which is centromeric to the FHIT gene, showed the presence of the target sequences. Fluorescence in situ hybridization analysis using probes covering exons 5 and 8 revealed no difference in the signal patterns between leukemia and normal cells, showing one or two signal doublets in more than 90% of nuclei, and indicated that gross segments of the FHIT gene were not homozygously deleted in these cases. A small number of transcripts with an aberrant fusion between exons 2 and 7 were detected by RT-PCR in the bone marrow cells from four healthy individuals. Granulocytes, lymphocytes, and monocytes in the bone marrow cells of a healthy individual contained transcripts with the same fusion. This unique fusion of exons 2 and 7 might be preferentially seen in either neoplastic or normal hematopoietic cells, regardless of their lineage. The finding that FHIT expression was abolished in the majority of leukemia cases might support the hypothesis that the FHIT gene acts as a tumor suppressor, at least in leukemia.

Acid Anhydride Hydrolases↗
PubMed2006

Development and evaluation of different normalization strategies for gene expression studies in Candida albicans biofilms by real-time PCR.

BACKGROUND: Candida albicans biofilms are commonly found on indwelling medical devices. However, the molecular basis of biofilm formation and development is not completely understood. Expression analysis of genes potentially involved in these processes, such as the ALS (Agglutinine Like Sequence) gene family can be performed using quantitative PCR (qPCR). In the present study, we investigated the expression stability of eight housekeeping genes potentially useful as reference genes to study gene expression in Candida albicans (C. albicans) biofilms, using the geNorm Visual Basic Application (VBA) for Microsoft Excel. To validate our normalization strategies we determined differences in ALS1 and ALS3 expression levels between C. albicans biofilm cells and their planktonic counterparts. RESULTS: The eight genes tested in this study are ranked according to their expression stability (from most stable to least stable) as follows: ACT1 (beta-actin)/PMA1 (adenosine triphosphatase), RIP (ubiquinol cytochrome-c reductase complex component), RPP2B (cytosolic ribosomal acidic protein P2B), LSC2 (succinyl-CoA synthetase beta-subunit fragment), IMH3 (inosine-5'-monophosphate dehydrogenase fragment), CPA1 (carbamoyl-phosphate synthethase small subunit) and GAPDH (glyceraldehyde-3-phosphate dehydrogenase). Our data indicate that five genes are necessary for accurate and reliable normalization of gene expression data in C. albicans biofilms. Using different normalization strategies, we found a significant upregulation of the ALS1 gene and downregulation of the ALS3 gene in C. albicans biofilms grown on silicone disks in a continous flow system, the CDC reactor (Centre for Disease Control), for 24 hours. CONCLUSION: In conclusion, we recommend the use of the geometric mean of the relative expression values from the five housekeeping genes (ACT1, PMA1, RIP, RPP2B and LSC2) for normalization, when analysing differences in gene expression levels between C. albicans biofilm cells and planktonic cells. Validation of the normalization strategies described above showed that the ALS1 gene is overexpressed and the ALS3 gene is underexpressed in C. albicans biofilms grown on silicone in the CDC reactor for 24 hours.

Biofilms↗
PubMed2006

Extra-long PCR, an identifier of DNA adducts in single nematodes (Caenorhabditis elegans).

DNA adducts are frequently caused by chemical induced changes in DNA. If mis-repaired, they can lead to nucleotide substitutions, deletions or chromosomal rearrangements. Depending on adduct stereochemistry and properties of the DNA target, adducts can inhibit transcriptional mechanisms. Here we demonstrate how this phenomenon can be exploited to detect DNA adducts in individual nematodes (Caenorhabditis elegans). An extra-long (XL)-PCR (16,144 bp) target amplicon, the 11 exon spanning ced-1, could be amplified reliably from genomic lysate extracted from single nematodes. Amplification efficiency was assessed by means of a second, fully quantitative PCR. Following the normalization with an invariant control gene, adduct formation could be evaluated by the identification of XL-PCR amplifications that were, relative to the control gene, reduced or inhibited by >95%. No DNA adducts could be detected in C. elegans maintained under optimal growth conditions (no exposure controls) or nematodes exposed to 20 microg/g copper sulfate (exposure negative control). However, exposure to 5 mug/g benzo[a]pyrene induced a stark response, with 40% of nematodes displaying measurable DNA adducts. Similarly, adducts were identified in 10% of nematodes subjected to 3 microg/g fluoranthene or a mixture containing 0.5 microg/g benzo[a]pyrene and 1 microg/g fluoranthene.

Animals↗
PubMed1994

Genetic changes: relevance for diagnosis and detection of minimal residual disease in acute lymphoblastic leukaemia.

Cure can now be achieved in a proportion of patients with ALL. However, relapse and eventual treatment failure occur in many cases receiving identical treatment, presumably as a result of failure to eradicate MRD. While for many years marrow morphology has been the standard by which leukaemic remission has been assessed, more sensitive techniques have been developed for detection of MRD including immunophenotypic analysis, and as discussed in this chapter, methods which detect leukemia-associated clonal genetic changes at the karyotypic and genomic levels. Table 10 lists the applicability and sensitivity of various markers used in MRD analysis in ALL. It is apparent that of the karyotypic and molecular approaches described, only PCR-based strategies for detection of either leukaemia-specific translocations or clonal Ag receptor rearrangements are reliably applicable to a high proportion of both B- and T-ALL at sufficiently high sensitivity. Initial clinical studies of patients undergoing therapy for ALL using a variety of PCR-based methods suggest that in some cases a persistent or increasing level of residual disease may be predictive for clinical relapse, although a number of technical factors and the phenomena of oligo-clonality and clonal evolution may limit the usefulness of this analysis in a few instances. From current available data it appears that in order to define the potential predictive value of PCR detection of MRD a large number of patients will need to be prospectively assessed over several years at multiple time points during and after therapy, preferably using more than one semi-quantitative PCR approach. In addition to reliable prediction of clinical relapse allowing appropriate individual treatment modification, progress in the molecular detection of MRD in ALL is also likely to be of benefit in the assessment of the efficacy of autograft purging and the evaluation of new therapeutic strategies such as the use of biological response modifiers to eliminate a low tumour burden.

Adolescent↗
PubMed2006

Transforming growth factor-beta expression induced by rhinovirus infection in respiratory epithelial cells.

Rhinovirus infection of the lower airways is now a recognized disease, associated with bronchiolitis and asthma. The bronchial epithelial cells are the host cells when rhinovirus infection occurs in the airway. It was hypothesized that a pro-fibrotic growth factor response may occur in these infected cells, leading to production of a key transforming growth factor, TGF-beta-1. Bronchial epithelial cells were inoculated with human rhinovirus and compared at day 1, 3 and 5 to control non-infected cells. Cell culture supernatant fluid and cellular RNA were isolated. The amount of released TGF-beta protein was measured by enzyme-linked immunosorbent assay (ELISA). Expression of TGF-beta at the level of transcription was measured by polymerase chain reaction (PCR) and gel electrophoresis. The results show that at all time points studied, TGF-beta production is greater in the infected cells, as demonstrated by ELISA (P<0.05) and by semi-quantitative PCR analysis. It was concluded that bronchial epithelial cells infected with common cold virus and rhinovirus, showed higher levels of TGF-beta. The production of TGF-beta may be indicative of a normal repair mechanism to counter inflammation, or in the setting of persistent asthma, could potentially lead to increased fibrosis and collagen deposition.

Cells, Cultured↗
PubMed2004

Effects of feeding a probiotic preparation (SIM) containing inulin on the severity of colitis and on the composition of the intestinal microflora in HLA-B27 transgenic rats.

An overly aggressive immune response to the intestinal microflora in a genetically susceptible host background has been implicated in the pathogenesis of inflammatory bowel diseases. We measured the impact of a probiotic preparation (SIM) containing inulin on the severity of colitis and on intestinal microflora profiles of HLA-B27-beta(2)-microglobulin transgenic (TG) rats. SIM is a mixture of lactobacilli, bifidobacteria, and inulin. Two-month-old TG rats received either SIM or water. Control TG rats received metronidazole, alone or in combination with SIM, for 8 weeks. Nontransgenic rats received SIM or water. The cecal content was removed for analysis of the intestinal microflora by PCR combined with denaturing gradient gel electrophoresis. The colon was scored for histological evidence of inflammation, colonic myeloperoxidase activity and interleukin-1beta RNA levels were measured photometrically or by real-time quantitative PCR. At 4 months, the colonic inflammation of TG rats treated with SIM was histologically diminished compared to that in untreated TG rats (2.2 +/- 0.2 versus 2.9 +/- 0.1; P </= 0.03). The administration of SIM altered the microflora profiles of TG rats by increasing the diversity and stimulating specifically the growth of Bifidobacterium animalis. The probiotic bacteria added to SIM were below the detection level in cecal stool samples at the end of the study period. The administration of SIM resulted in a measurable impact on the cecal microflora profiles of TG rats with attenuation of colitis. The lack of detection of any added probiotic bacteria in the cecal content suggests that prebiotic inulin is the major effective compound.

Animals↗
PubMed2003

Evaluation of a rapid, quantitative real-time PCR method for enumeration of pathogenic Candida cells in water.

Quantitative PCR (QPCR) technology, incorporating fluorigenic 5' nuclease (TaqMan) chemistry, was utilized for the specific detection and quantification of six pathogenic species of Candida (C. albicans, C. tropicalis, C. krusei, C. parapsilosis, C. glabrata and C. lusitaniae) in water. Known numbers of target cells were added to distilled and tap water samples, filtered, and disrupted directly on the membranes for recovery of DNA for QPCR analysis. The assay's sensitivities were between one and three cells per filter. The accuracy of the cell estimates was between 50 and 200% of their true value (95% confidence level). In similar tests with surface water samples, the presence of PCR inhibitory compounds necessitated further purification and/or dilution of the DNA extracts, with resultant reductions in sensitivity but generally not in quantitative accuracy. Analyses of a series of freshwater samples collected from a recreational beach showed positive correlations between the QPCR results and colony counts of the corresponding target species. Positive correlations were also seen between the cell quantities of the target Candida species detected in these analyses and colony counts of Enterococcus organisms. With a combined sample processing and analysis time of less than 4 h, this method shows great promise as a tool for rapidly assessing potential exposures to waterborne pathogenic Candida species from drinking and recreational waters and may have applications in the detection of fecal pollution.

Bathing Beaches↗
PubMed2003

Real-time PCR for simultaneous detection and quantification of Borrelia burgdorferi in field-collected Ixodes scapularis ticks from the Northeastern United States.

The density of spirochetes in field-collected or experimentally infected ticks is estimated mainly by assays based on microscopy. In this study, a real-time quantitative PCR (qPCR) protocol targeting the Borrelia burgdorferi-specific recA gene was adapted for use with a Lightcycler for rapid detection and quantification of the Lyme disease spirochete, B. burgdorferi, in field-collected Ixodes scapularis ticks. The sensitivity of qPCR for detection of B. burgdorferi DNA in infected ticks was comparable to that of a well-established nested PCR targeting the 16S-23S rRNA spacer. Of the 498 I. scapularis ticks collected from four northeastern states (Rhode Island, Connecticut, New York, and New Jersey), 91 of 438 (20.7%) nymphal ticks and 15 of 60 (25.0%) adult ticks were positive by qPCR assay. The number of spirochetes in individual ticks varied from 25 to 197,200 with a mean of 1,964 spirochetes per nymphal tick and a mean of 5,351 spirochetes per adult tick. No significant differences were found in the mean numbers of spirochetes counted either in nymphal ticks collected at different locations in these four states (P = 0.23 by one-way analysis of variance test) or in ticks infected with the three distinct ribosomal spacer restriction fragment length polymorphism types of B. burgdorferi (P = 0.39). A high degree of spirochete aggregation among infected ticks (variance-to-mean ratio of 24,877; moment estimate of k = 0.279) was observed. From the frequency distribution data and previously published transmission studies, we estimated that a minimum of 300 organisms may be required in a host-seeking nymphal tick to be able to transmit infection to mice while feeding on mice. These data indicate that real-time qPCR is a reliable approach for simultaneous detection and quantification of B. burgdorferi infection in field-collected ticks and can be used for ecological and epidemiological surveillance of Lyme disease spirochetes.

Animals↗
PubMed1998

Quantitative reverse transcriptase PCR to gauge increased protease-activated receptor 1 (PAR-1) mRNA copy numbers in the Wobbler mutant mouse.

Thrombin acts on cells through the surface protease-activated receptor 1 (PAR-1), a G-protein-coupled member of the seven-transmembrane domain superfamily. On neural cells, thrombin has deleterious effects, killing neurons through apoptosis. Consequently, knowledge of PAR-1 expression in the nervous system may help to elucidate the role of thrombin in neurodegenerative disease. We developed a mimic construct to facilitate the highly sensitive technique of quantitative reverse transcriptase to PCR (qRT-PCR) to measure the differential expression of low copy number PAR-1 mRNA in neurodegenerative model systems. In this article, we report our results comparing homozygous wobbler (wr/wr) mice and normal littermates. By optimizing the transcription and quantitative PCR procedures to facilitate rapid copy number determination in small RNA samples, we documented a fivefold greater level of PAR-1 mRNA in the cervical spinal cord of wr/wr.

Animals↗
PubMed2005

Identification and characterization of three large deletions and a deletion/polymorphism in the CFTR gene.

Cystic fibrosis (CF) is mainly caused by small molecular lesions of the CFTR gene; mutation detection methods based on conventional PCR do not allow the identification of all CF alleles in a population and large deletions may account for a number of these unidentified molecular lesions. It is only recently that the availability of quantitative PCR methodologies made the search for large gene rearrangements easier in autosomal diseases. Using a combination of different methods, nine of the 37 unidentified CF alleles (24%) were found to harbor large deletions in our cohort of 1600 CF alleles. Three are new deletions, and we report the breakpoints of the previously described EX4_EX10del40kb deletion. An intronic deletion polymorphism affecting intron 17b was also found on almost 1% of "normal" chromosomes. Examination of the breakpoint sequences confirmed that intron 17b is indeed a hot spot for deletions, and that most of these rearrangements are caused by non-homologous recombination.

Adolescent↗