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PubMed2006

A real time PCR assay for the detection and quantification of orf virus.

A real time quantitative PCR assay based on TaqMan technology was developed for orf virus (ORFV) DNA quantification in clinical samples, infected cells and organotypic cultures. This method was based on the amplification of a 70 bp fragment from the ORFV B2L gene (orthologue of the Vaccinia virus Copenhagen F13L gene) that encodes the major envelope protein. Both intra- and inter-assay variability were well within +/-0.25 log(10) S.D. showing the high efficiency and reproducibility of the assay. The TaqMan PCR was subsequently used to determine the titre of several batches of the ORFV strain NZ-2, with it being possible to quantify virus solutions in the range of 1 x 10(1) to 1 x 10(6) TCID(50)/ml. A good correlation between the titre determined by the TaqMan PCR and by conventional endpoint dilution was found. The PCR assay is reproducible and can be used for a rapid quantification of ORFV in vitro and ex vivo, being readily achievable within 1h.

Animals↗
PubMed2007

Genome-wide oligonucleotide-based array comparative genome hybridization analysis of non-isolated congenital diaphragmatic hernia.

Non-isolated congenital diaphragmatic hernia (CDH+) is a severe birth defect that is often caused by de novo chromosomal anomalies. In this report, we use genome-wide oligonucleotide-based array comparative genome hybridization (aCGH) followed by rapid real-time quantitative PCR analysis to identify, confirm and map chromosomal anomalies in a cohort of 26 CDH+ patients. One hundred and five putative copy number changes were identified by aCGH in our cohort of CDH+ patients. Sixty-one of these changes (58%) had been previously described in normal controls. Twenty of the remaining 44 changes (45%) were confirmed by quantitative real-time PCR or standard cytogenetic techniques. These changes included de novo chromosomal abnormalities in five of the 26 patients (19%), two of whom had previously normal G-banded chromosome analyses. Data from these patients provide evidence for the existence of CDH-related genes on chromosomes 2q37, 6p22-25 and 14q, and refine the CDH minimal deleted region on 15q26 to an interval that contains COUP-TFII and only eight other known genes. Although COUP-TFII is likely to play a role in the development of CDH in patients with 15q26 deletions, we did not find COUP-TFII mutations in 73 CDH samples. We conclude that the combination of oligonucleotide-based aCGH and quantitative real-time PCR is an effective method of identifying, confirming and mapping clinically relevant copy number changes in patients with CDH+. This method is more sensitive than G-banded chromosome analysis and may find wide application in screening patients with congenital anomalies.

Chromosome Aberrations↗
PubMed2005

Development of quantitative reverse transcriptase PCR assays for measuring gene expression.

Real-time, quantitative reverse transcriptase (RT)-PCR is a very useful and powerful technology for analysis of gene expression. At a first pass, real-time PCR appears to be a simple extension of regular PCR, and it should therefore be easy for an experienced PCR user to convert to quantitative assays. In practice, however, our experience would indicate that this is not usually the case, and most novice real-time PCR users run into problems even though they are very capable at regular PCR. One problem is that, unlike Northern blots, which are technically difficult but typically either work or do not, real-time PCR assays, even poorly designed ones, usually give data. Unfortunately, these data, or their interpretation, may be erroneous, since there are many potential pitfalls that need to be avoided when designing and using real-time PCR for measurement of gene expression. The purpose of this chapter is not to try to discuss the complexities of real-time PCR in detail (which would require a whole book), but, instead, to provide a simple outline for the development of real-time PCR assays. If followed, these guidelines should allow the reader to develop real-time PCR assays that avoid the most common pitfalls and that are capable of producing reliable and accurate gene expression data.

Animals↗
PubMed2001

Gene expression of glucose transporters and its regulation by glucose in mesothelial cells.

OBJECTIVE: To observe the influence of glucose on the expression of glucose transporters (GluTs) in peritoneal tissues. METHODS: Mesothelial cells (MsCs) from Sprague-Dawley (SD) rats were cultured in medium with glucose 214.4 mmol/L or 75.5 mmol/L. The normal medium with glucose 17.5 mmol/L was used as control. Total RNA was extracted from each sample after 24 hours incubation. Reverse transcript polymerase chain reaction (RT-PCR) was performed with primers corresponding to sodium-glucose transporter (SGIT1) and GluT1-GluT4. mRNA expression of the above GluTs from each sample was measured with quantitative PCR. RESULTS: GluT1 and GluT2 mRNA can be detected in MsCs from SD rats, while no positive bands can be found specifically for GluT3, GluT4 and SGiT1. Quantitating the amount of PCR products indicated that the levels of GluT1 mRNA in MsCs cultured 24 h in both 214.4 mmol/L glucose and 75.5 mmol/L glucose medium decreased dramatically compared with that in normal medium (P < or = 0.01). While under the same conditions, the levels of GluT2 mRNA in MsCs cultured 24 h in 214.4 mmol/L and 75.5 mmol/L glucose medium both increased significantly (P < 0.01). CONCLUSIONS: GluT1 is strongly expressed in MsCs under normal glucose levels and decreased dramatically under high glucose conditions, while GluT2 expressed at a low level in normal medium and increased greatly after incubation in high glucose conditions. This may play a great role in glucose absorption during peritoneal dialysis and have some connection with ultrafiltration failure due to the alteration of glucose absorption after long-term dialysis.

Animals↗
PubMed2004

DNA microarray allows molecular profiling of rheumatoid arthritis and identification of pathophysiological targets.

This study was undertaken to evaluate the possibility to obtain a molecular signature of rheumatoid arthritis (RA) comparatively osteoarthritis (OA), and to lay the bases to develop new diagnostic tools and identify new targets. Microarray technology was used for such an analysis. The gene expression profiles of synovial tissues from patients with confirmed RA, and patients with OA were established and compared. A set of 63 genes was selected, based, more specifically, on their overexpression or underexpression in RA samples compared to OA. Results for six of these genes have been verified by quantitative PCR using both samples identical to those used in the microarray experiments and entirely separate samples. Expression profile of the 48 known genes allowed the correct classification of additional RA and OA patients. Furthermore, the distinct expression of three of the selected genes was also studied by quantitative RT-PCR in cultured synovial cells. Detailed analysis of the expression profile of the selected genes provided evidence for dysregulated biological pathways, pointed out to chromosomal location and revealed novel genes potentially involved in RA. It is proposed that such an approach allows valuable diagnosis/prognostics tools in RA to be established and potential targets for combating the disease to be identified.

Adult↗
PubMed2004

Transcriptional silencing is associated with extensive methylation of the CMV promoter following adenoviral gene delivery to muscle.

BACKGROUND: Although the transient nature of transgene expression using first-generation adenovirus (Ad) vectors is well known, the exact mechanisms responsible for this phenomenon are uncertain. METHODS: Rats were given intramuscular (i.m.) injections of a first-generation Ad containing the human fibroblast growth factor 4 (hFGF-4) gene driven by the cytomegalovirus (CMV) promoter and enhancer (CMV-PE). The copy number of hFGF-4 mRNA and viral DNA was measured in the same muscles by quantitative RT-PCR and quantitative PCR at times between 1 h and 84 days after virus injection. Quantitative Southern blot analysis for the intact hFGF-4 transcription unit DNA was also performed, and the methylation status of the CMV-PE DNA in the muscle was determined using bisulfite sequencing. RESULTS: The copy number of hFGF-4 mRNA peaked at 6 h then decreased 56-fold by 24 h, and a further 240-fold between days 3 and 28. Although the viral DNA copy number also decreased 23-fold between 6 h and 28 days, the ratio of copies of hFGF-4 mRNA per copy of viral DNA decreased 385-fold over this period. Methylation of the CMV-PE DNA in the muscle at both CpG and non-CpG sites was observed 24 h after virus administration and had increased at day 7. CONCLUSIONS: Decreased transcription associated with extensive methylation of the CMV-PE was the major mechanism responsible for the decrease in transgene mRNA levels. Strategies for preventing transcriptional silencing will be valuable for improving the duration of transgene expression from adenoviral vectors.

Animals↗
PubMed2006

Expression and quantification of LYVE-1 in human colorectal cancer.

The recent discovery of a new hyaluronan (HA) receptor, LYVE-1 (lymphatic vessel endothelial HA receptor), has been received with great interest regarding its specific expression in the lymphatic system. The process of lymphangiogenesis or the formation of new lymphatics in tumours is important because it serves as a major route for cancer metastasis. Therefore, methods to quantify lymphangiogenesis by measuring LYVE-1 have been studied extensively in searching for its possible role in cancer diagnosis, prognosis and even targeted treatment of lymphatic tumour metastasis. Here we report a quantitation study on lymphangiogenesis by either quantitative PCR or immunohistochemistry approaches in detecting LYVE-1 expression in human colorectal tumour. Real-time quantitative polymerase chain reaction (RTQ-PCR) was carried out to quantify LYVE-1 levels in colorectal cancer samples. Also, the same specimen was observed for LYVE-1 expression by immunohistochemical stain. By RTQ-PCR amplification, LYVE-1 was highly expressed in colorectal specimens and LYVE-1 signal from non-cancer tissue of normal control was much weaker by conventional RTPCR. Immunohistochemical stain showed that LYVE-1 was significantly expressed in cancer tissues (especially in the margin region of cancer), whereas in non-cancer specimens fewer positive stains were revealed. The results suggested that the LYVE-1 molecule was expressed significantly in colorectal specimens, which may imply a new marker for a malignant situation.

Base Sequence↗
PubMed2004

Design and performance testing of quantitative real time PCR assays for influenza A and B viral load measurement.

BACKGROUND: The antiviral effect of anti-influenza drugs such as zanamivir may be demonstrated in patients as an increased rate of decline in viral load over a time course of treatment as compared with placebo. Historically this was measured using plaque assays, or Culture Enhanced Enzyme Linked Immunosorbent Assay (CE-ELISA). OBJECTIVES: to develop and characterise real time quantitative PCR (qPCR) assays to measure influenza A and B viral load in clinical samples, that offer improvements over existing methods, in particular virus infectivity assays. STUDY DESIGN: The dynamic range and robustness were established for the real time qPCR assays along with stability of the assay components. Cross validation of the real time PCR assays with CE-ELISA was performed by parallel testing of both serial dilutions of three different subtypes of cultured virus and a panel of influenza positive throat swab specimens. RESULTS: the assays were specific for influenza A and B and the dynamic ranges were at least seven logs. The assay variability was within acceptable limits but increased towards the lower limit of quantification, which was 3.33 log(10) viral cDNA copies/ml of virus transport medium (ten viral RNA copies/PCR). The components of the assay were robust enough to withstand extended storage and several freeze-thaw cycles. For the real time PCR assays the limit of quantification was equivalent to the virus infectivity cut off, which equates to a 93-fold increase in sensitivity. CONCLUSION: Well characterised real time PCR assays offer significant improvements over the existing methods for measuring the viral load of strains of influenza A and B in clinical specimens.

DNA, Complementary↗
PubMed1996

Use of a fluorescent-PCR reaction to detect genomic sequence copy number and transcriptional abundance.

We present a fluorescent-PCR-based technique to assay genomic sequence copy number and transcriptional abundance. This technique relies on the ability to follow fluorescent PCR progressively in real time during the exponential phase of the reaction so that quantitative PCR is accomplished. We demonstrated the ability of this technique to quantitate both known deletions and amplifications of loci that have been measured previously by other methods, and to measure transcriptional abundance. Using an efficient variant of the fluorescent-PCR technology, we can monitor transcription semiquantitatively. The ability to detect all amplifications and deletions at any single copy locus by PCR makes this the technique of choice to assay genomic sequence copy number anomalies in birth defects and cancers. The ability to detect variations in transcript abundance enables this technique to fashion a time and tissue analysis of transcription.

Chromosome Aberrations↗
PubMed2005

Highly specific overexpression of the transcription factor SOX11 in human malignant gliomas.

Malignant glioma comprises the majority of primary human brain tumors with 16,800 new cases reported each year in the USA. Its prognosis remains dismal despite numerous attempts to improve conventional therapeutic modalities. Therefore, much effort is devoted to the exploration of alternative forms of treatment such as immunotherapy. The identification of potential target structures highly overexpressed in brain tumors is a crucial prerequisite for the activation of the immune defense against malignant glioma cells. By screening an expression database for genes highly expressed in glioblastoma multiforme (GBM), we identified the Pit-Oct-Unc (POU) cooperating transcription factor SOX11 that is known to be crucially involved in brain development. Analysis of the expression pattern of SOX11 in different normal adult and fetal tissues by multiple tissue dot blot and by a highly sensitive quantitative PCR assay confirmed the selective overexpression of SOX11 in fetal brain tissue. Examination of tissue specimens obtained from malignant gliomas and from normal brain by quantitative real-time PCR (Q-RT-PCR) revealed upregulation of SOX11 in almost all tumor samples (15/16) as compared to the pooled normal brain. Seventy-five percent of the tumor samples (12/16) showed a 5- to more than 600-fold overexpression. We conclude that, after downregulation of SOX11 in the adult brain, its expression is reactivated during tumorigenesis and that SOX11 therefore represents a promising novel molecular target for adjuvant therapy of malignant gliomas.

Brain Neoplasms↗
PubMed2002

Calibrated real-time PCR assay for quantitation of human herpesvirus 8 DNA in biological fluids.

Accurate laboratory tests for the diagnosis of active human herpesvirus 8 (HHV-8) infection are becoming essential to study the pathogenesis of HHV-8-associated tumors and for the clinical management of HHV-8-infected individuals. We have developed a highly sensitive, calibrated quantitative real-time PCR assay for the measurement of cell-free HHV-8 DNA in body fluids, based on the addition of a synthetic DNA calibrator prior to DNA extraction. The calibrator controls each sample for the presence of PCR inhibitors, determines a cutoff value of sensitivity for negative samples, and normalizes positive samples for the efficiency of DNA recovery. The assay shows a wide dynamic range of detection (between 1 and 10(6) viral genome equivalents/reaction) and a high degree of accuracy even in the presence of high amounts (up to 1 micro g) of human genomic DNA. Moreover, the assay has a very high sensitivity (lower detection limit, 10 genome equivalents/ml) and a high degree of reproducibility and repeatability with a coefficient of variation (CV) of <15 and 23%, respectively. Furthermore, the use of the calibrator improves the accuracy of quantitation and decreases the intersample variability (CV, 9 and 6%, respectively). The sensitivity and specificity of the assay were tested with a series of clinical specimens obtained from patients affected by various HHV-8-related diseases, as well as from a wide number of controls. In conclusion, our calibrated real-time PCR assay provides a reliable high-throughput method for quantitation of HHV-8 DNA in clinical and laboratory specimens.

AIDS-Related Opportunistic Infections↗
PubMed1999

Development of a chemiluminescence competitive PCR for the detection and quantification of parvovirus B19 DNA using a microplate luminometer.

BACKGROUND: Quantitative PCR of viral nucleic acids can be useful clinically in diagnosis, risk assessment, and monitoring of antiviral therapy. We wished to develop a chemiluminescence competitive PCR (cPCR) for parvovirus B19. METHODS: Parvovirus DNA target sequences and competitor sequences were coamplified and directly labeled. Amplified products were then separately hybridized by specific biotin-labeled probes, captured onto streptavidin-coated ELISA microplates, and detected immunoenzymatically using chemiluminescent substrates of peroxidase. Chemiluminescent signals were quantitatively analyzed by a microplate luminometer and were correlated to the amounts of amplified products. RESULTS: Luminol-based systems displayed constant emission but had a higher detection limit (100-1000 genome copies) than the acridan-based system (20 genome copies). The detection limit of chemiluminescent substrates was lower (20 genome copies) than colorimetric substrates (50 genome copies). In chemiluminescence cPCR, the titration curves showed linear correlation above 100 target genome copies. Chemiluminescence cPCR was positive in six serum samples from patients with parvovirus infections and negative in six control sera. CONCLUSIONS: The chemiluminescence cPCR appears to be a sensitive and specific method for the quantitative detection of viral DNAs.

DNA, Viral↗
PubMed2005

Comparison of six methods of extracting Mycobacterium tuberculosis DNA from processed sputum for testing by quantitative real-time PCR.

Six methods of extracting Mycobacterium tuberculosis DNA from sputum for testing by quantitative PCR were compared: Tris-EDTA (TE) buffer, PrepMan Ultra, 2% sodium dodecyl sulfate (SDS)-10% Triton X with and without sonication, Infectio Diagnostics, Inc. (IDI) lysing tubes, and QIAGEN QIAamp DNA mini kit; all included a 15-min boiling step. Pooled digested and decontaminated sputum was spiked with M. tuberculosis ATCC 27294. Each extraction method was repeated eight times. Quantitative PCR was performed on the Smart Cycler and Rotor-Gene 3000 using primers targeting an 83-bp fragment of IS6110. An minor grove binding Eclipse probe with a fluorescent label was used for detection. An internal control was included to detect amplification inhibition. The limit of detection of M. tuberculosis DNA was 0.5 fg with both instruments. Calculated DNA concentrations (picograms) extracted using IDI, PrepMan, QIAGEN, and TE were 42.8, 30.4, 28.2, and 7.4, respectively, when run on the Smart Cycler, and 51.7, 20.1, 14.9, and 8.6, respectively, when run on Rotor-Gene. All extractions using SDS/Triton X with or without sonication were inhibited. Of the extraction methods evaluated, IDI lysis tubes provided the greatest yield of mycobacterial DNA, and the procedure can be completed in less than 1 h versus 2.5-3 h for the QIAGEN extraction.

Base Sequence↗
PubMed2005

Prevalence of hepatitis C infection in a population of asymptomatic people in a checkup unit in Mexico city.

Hepatitis C virus (HCV) infection has an estimated prevalence of 3% around the world. Unfortunately, many persons with HCV infection are asymptomatic. The aim of this study was to determine the prevalence of hepatitis C Virus infection in asymptomatic persons. This study was carried out in a population-based cross-sectional study in the Checkup Unit of University Hospital in Mexico City. Patients with two or more HCV risk factors were studied. Serum specimens from all patients were screened for HCV RNA by qualitative polymerase chain reaction (PCR). HCV RNA-positive serum was also screened by quantitative PCR and for HCV genotype. Three hundred asymptomatic people were included, 194 men and 106 women, with a mean age of 46.8+/-11.9. Six (2%) of the 300 people were positive and viremic. The most common risk factors in positive patients were manicures or pedicures with a nonpersonal instrument and more than three sexual partners. We concluded that hepatitis C is frequent in asymptomatic people, and those people are often viremic. In addition, this study suggests that the spectrum of liver disease in asymptomatic and newly diagnosed HCV-positive persons is broad.

Adult↗
PubMed2004

Highly specific marker genes for detecting minimal gastric cancer cells in cytology negative peritoneal washings.

Peritoneal wash cytology plays a pivotal role in the decision for gastric cancer treatment because advanced gastric cancer often turns out incurable with peritoneal metastasis. Molecular detection of minimal cancer cells from peritoneal washings may overcome the sensitivity boundary of conventional cytology and contribute to the prediction of the disease outcome. To select marker candidates out of ten thousands of genes, we performed microarray analyses in 12 gastric cell lines and 8 peritoneal washings of early stage cases. With 40 candidates selected by the above expression profiling, RT-PCR in 16 representative peritoneal wash samples was performed to identify genes specific to cytology positive samples. The finally selected five genes, CK20, FABP1, MUC2, TFF1, and TFF2, were then evaluated for their utility as a marker for minimal residual disease in 99 peritoneal wash samples. Nested RT-PCR using the five genes showed positive results highly specific to incurable cases (91-100%). With a high specificity, the combination of these five genes succeeded in identifying 6 out of 20 (30%) additional patients with all types of early recurrence that could not be predicted by the conventional method. The six newly identified recurrences included four non-peritoneal ones, showing that RT-PCR using the five genes without a real-time quantitative PCR technique contributes to the detection of minimal residual disease.

Ascitic Fluid↗
PubMed2006

Human herpesvirus-6B active infection associated with relapsing bilateral anterior optic neuritis.

BACKGROUND AND OBJECTIVES: Human herpesvirus-6 (HHV-6) is the causative agent of exanthem subitum. Both HHV-6 variants, A and B, have been associated with central nervous system (CNS) diseases, suggesting a wide neuropathogenic potential. We describe a case of recurrent bilateral anterior optic neuritis with HHV-6 active infection associated with clinical relapses. CASE REPORT: A 23-year old woman presented with progressive visual impairment, bilateral papillitis and painful ocular movements. Nested polymerase chain reaction (PCR) for DNA viruses, HHV-6 variant specific real time quantitative PCR, serological analysis and retrotranscription PCR (RT-PCR) for HHV-6 mRNA transcripts were performed. Nested PCR in PBMC and CSF samples was negative for all viruses but positive for HHV-6 DNA, subtyped as HHV-6B. The disease had a relapsing/remitting course. During relapses PBMC samples remained positive for HHV-6 DNA, and HHV-6 active infection was confirmed by the presence of anti-HHV-6 IgM and of HHV-6 U27 mRNA transcript. High viremia levels and relapses were overlapping. After the last relapse, the patient was successfully treated with gancyclovir. CONCLUSIONS: The case reported here suggests a possible association of HHV-6 in bilateral optic neuritis. HHV-6 could be monitored when bilateral optic neuritis is identified, in order to establish an appropriate antiviral therapy.

Adult↗
PubMed2001

Parvovirus B19 DNA in plasma pools and plasma derivatives.

BACKGROUND AND OBJECTIVES: Human parvovirus B19 (B19) has been transmitted by various plasma-derived medicinal products. The aim of this study was to determine the frequency and the level of B19 DNA contamination in plasma pools destined for fractionation and in a broad range of plasma derivatives. In addition, removal of B19 DNA by the manufacturing process was investigated in cases where corresponding samples from plasma pool and product were available. MATERIALS AND METHODS: Plasma pool samples and blood products were tested for B19 DNA by nested polymerase chain reaction (PCR), and the viral DNA content was determined by TaqMan quantitative PCR. RESULTS: Two-hundred and twenty two of 372 plasma pools for fractionation contained B19 DNA at concentrations of 10(2)-10(8) genome equivalents/ml (geq/ml). While approximately 65% of the DNA-positive plasma pools were only moderately contaminated (< 10(5) geq/ml), 35% contained > 10(6) geq/ml. High frequencies of contamination were detected in Factor VIII (79 of 91), prothrombin complex concentrates (38 of 43) and Factor IX (41 of 62), where the concentration of B19 DNA ranged between 102 and 107 geq/ml. A lower level of B19 DNA contamination was found in antithrombin III (five of 26 samples), in anti-D immunoglobulins (three of 37 samples) and in albumin (four of 51 samples), with levels ranging between 10(2) and 10(3) geq/ml. Furthermore, investigation of plasma pools for solvent/detergent plasma (S/D plasma), from two manufacturers, revealed B19 DNA in 15 of 66 batches at concentrations of 10(2)-10(8) geq/ml. Similar concentrations were detected in the corresponding final S/D plasma products. Anti-B19 immunoglobulin G (IgG) was found in plasma pools and S/D plasma at concentrations of approximately 40 IU/ml. CONCLUSION: Although positive PCR results do not necessarily reflect infectivity, these data show that B19 is a common contaminant in plasma pools and in plasma-derived medicinal products. Considering the resistance of animal parvoviruses to inactivation by heat and chemical agents, and the absence of specific information for B19, the risk of B19 transmission by plasma products should be considered. Physicians should be aware of this problem when treating patients of B19-related risk groups. The plasma fractionation industry should continue their efforts to avoid B19 contamination of plasma derivatives and develop methods which are effective in removing/inactivating parvovirus B19.

Blood↗
PubMed1994

An internally controlled virion PCR for the measurement of HIV-1 RNA in plasma.

We have developed an assay to measure the HIV-1 RNA in patients' plasma or sera using an infectious mutant virus as an internal control. The mutant virus VX-46 has a 25-bp insert in a conserved region between the primer-binding and major splice donor sites. To utilize this virus as an internal control, different dilutions of this virus were added to aliquots of plasma sample to be measured, RNA was isolated and reverse-transcribed to cDNA. PCR was performed with primers selected to include the sequences on either side of the insert contained in the externally added virus. The DNA product from the control virus is 25 bp longer than that from the virus present in plasma. The amount of viral RNA present in a plasma sample is calculated after the PCR-amplified products are separated by gel electrophoresis. Unlike other quantitative PCR assays, this internally controlled virion PCR (ICVPCR) assay eliminates errors introduced by variable recovery during the RNA purification step, therefore, enhancing the accuracy of the assay.

Base Sequence↗