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PubMed1990

Measurement by quantitative PCR of changes in HPRT, PGK-1, PGK-2, APRT, MTase, and Zfy gene transcripts during mouse spermatogenesis.

A reverse transcriptase-polymerase chain reaction assay (RT-PCR) was used quantitatively to measure accumulated levels of RNA transcripts in total mouse RNAs derived from male germ cells at various spermatogenic stages. RNA levels for two X-linked enzymes, phosphoglycerate kinase (PGK-1) and hypoxanthine phosphoribosyl transferase (HPRT), both decrease during spermatogenesis, although the transcript levels decrease much more rapidly for PGK-1. RNA for the Y-linked ZFY (zinc finger protein) is elevated in all spermatogenic cell fractions tested, being particularly high in leptotene/zygotene spermatocytes and round spermatids. RNA for adenine phosphoribosyltransferase (APRT) increases 5-fold to a peak during late pachynema. RNA for PGK-2, undetectable in spermatogonial cells, increases at least 50-fold by the round spermatid stage. DNA (cytosine-5-)-methyltransferase (MTase) transcript levels are over an order of magnitude higher throughout spermatogenesis than in non-dividing liver cells.

Adenine Phosphoribosyltransferase↗

PubMed2005

Optimized real-time quantitative PCR measurement of male fetal DNA in maternal plasma.

BACKGROUND: Circulating fetal DNA (cfDNA) in maternal plasma has been measured to investigate its possible relationship with pregnancy-related disorders, including fetal trisomy 21 and preeclampsia. The circulating concentrations of single-copy fetal genes, however, are close to the detection limits of PCR methods. METHODS: We optimized a protocol for the real-time quantitative PCR amplification of the multicopy sequence DYS14 on the Y-chromosome. This was compared with an established real-time PCR assay for the single-copy SRY gene. RESULTS: By probit regression analysis, the measurements of male DNA by the DYS14 assay had a 10-fold lower detection limit (0.4 genome equivalents) than did measurements of SRY. For plasma samples from women in the first trimester of pregnancy, imprecision (CV) was 2%-22% when amplifying DYS14 compared with 26%-140% for SRY. CONCLUSIONS: The low copy numbers of fetal DNA in plasma of women in the first trimester of pregnancy cannot be measured precisely when targeting single-copy sequences. Better results are obtained by amplifying a sequence that is present in multiple copies per male genome.

Chromosomes, Human, Y↗

PubMed2005

Standardization of parvovirus B19 DNA extraction from serum and quantitative PCR.

Human parvovirus B19 (B19) is a newly emerging virus causing a wide spectrum of clinical conditions. The corner stone of diagnosis of acute B19 infection is demonstration of anti-B19 IgM antibodies and its DNA by PCR. Sera of patients infected with B19 acutely or persistently shows intense viraemia (up to 10(12) virus particles/ ml) but extraction of B19 DNA from serum is a problematic task. There is no single standardized, cost-effective in-house method, which can successfully extract DNA of B19 from serum samples and which has been subsequently tested repeatedly by many investigators over time. We describe here an efficient in-house method of extraction of B19 DNA from serum and quantitate extracted DNA by PCR. Firstly, a quantitative PCR was done using 3 microl of a cloned B19 DNA (33.3 pg/ml) mixed in 47 microl of sterile distilled water which were further diluted from 10(-1) up to 10(-7) to find the lower limit of DNA detection. To mimic human serum samples with known quantity of B19, 3 ml of cloned B19 DNA (33.3 pg/ml) were mixed with 47ml of fetal calf serum (FCS; free of B19) and were similarly log diluted from 10(-1) to 10(-7) in 50 ml volume. In-house method of DNA extraction from serum (FCS+B19 DNA) were then performed followed by quantitative PCR. In both the cases, we were able to detect B19 DNA up to 10(-4) dilution which contained 0.6 fg of B19 DNA corresponding to 12 B19 genome equivalents/PCR reaction or 2.4 x 10(2) genome quivalents/ml of serum. Further, the entire procedure was repeated two more times and identical results were obtained confirming its reproducibility. Using this in-house method we extracted and amplified B19 DNA successfully from sera of clinically suspected cases of B19 infection (data not shown). Compared to other studies, the sensitivity of our in-house method was found to be superior hence recommended for developing countries as commercial kits are too costly.

Animals↗

PubMed2006

Validation of endogenous reference genes for expression profiling of RAW264.7 cells infected with Mycobacterium avium subsp. paratuberculosis by quantitative PCR.

Reference genes are frequently used to normalize between different biological samples the levels of mRNA measured using quantitative PCR (qPCR). The expression level of many commonly used reference genes has been shown to vary between tissues or cells, or following exposure to various treatments including infection with microbes. The selection of an appropriate reference gene for an individual experiment is therefore a crucial step in the process of accurately determining changes in gene expression. For this purpose, we analyzed the expression of nine commonly used reference genes in a murine macrophage cell line, RAW264.7, for their potential use in the analysis of differential gene expression by quantitative polymerase chain reaction (qPCR) following experimental infection with Mycobacterium avium subsp. paratuberculosis. Only one of nine putative reference genes tested, casc3a, was found to be suitable, and combinations of two or more reference genes were disadvantageous. Based on data from the study, we recommend an approach for selection of reference genes, conducting assays with technical replicates in duplicate rather than triplicate, determining decision-limit quality control criteria for technical replicates and assessing the significance of gene expression fold differences using DeltaDeltaC(t) based on knowledge of the variation in the reference gene.

Actins↗

PubMed2000

Comparison of competitive and positive control-based PCR quantitative procedures coupled with end point detection.

Two PCR methods using internal standards, coupled with our sandwich nonisotopic enzyme-linked oligosorbent assay (ELOSA) in microtiter plate format, were developed for quantitation of human immunodeficiency virus type 1 (HIV-1) provirus. We present an overview of both methodologies focusing on two major features, i.e., the conditions of equivalency of replication efficiency and the definition of criteria of acceptance validating a result. Quantitative competitive PCR (QC-PCR) was based on the coamplification of the HIV-1 nef gene with different amounts of a pNEFmut plasmid that contains the nef gene with different amounts of a pNEFmut plasmid that contains the nef region but with mutations in the capture probe recognition region. The NEF wild-type (NEF) and the NEF mimic (NEFmut) amplification products were differentiated in ELOSA. NEFmut OD to NEF OD ratios were plotted against the number of mimic copies, and the deduced linear curve permitted quantitation of HIV-1 copy number. Internally controlled PCR (IC-PCR) was based on coamplification of the HIV-1 nef gene with an internal endogenous standard, the ras gene, as a positive control of amplification. HIV-1 copy number was determined using external standard of known amounts of HIV-1 DNA. We address the advantages as well as the limitations of individuals protocols and discuss future improvements of quantitative amplification process.

DNA, Viral↗

PubMed2002

Detection of minute virus of mice using real time quantitative PCR in assessment of virus clearance during the purification of Mammalian cell substrate derived biotherapeutics.

A real time quantitative PCR assay has been developed for detecting minute virus of mice (MVM). This assay directly quantifies PCR product by monitoring the increase of fluorescence intensity emitted during enzymatic hydrolysis of an oligonucleotide probe labelled covalently with fluorescent reporting and quenching dyes via Taq polymerase 5'-->3' exonuclease activity. The quantity of MVM DNA molecules in the samples was determined using a known amount of MVM standard control DNA fragment cloned into a plasmid (pCR-MVM). We have demonstrated that MVM TaqMan PCR assay is approximately 1000-fold more sensitive than the microplate infectivity assay with the lowest detection limit of approximately one particle per reaction. The reliable detection range is within 100 to 10(9) molecules per reaction with high reproducibility. The intra assay variation is <2.5%, and the inter assays variation is <6.5% when samples contain >100 particles/assay. When we applied the TaqMan PCR to MVM clearance studies done by column chromatography or normal flow viral filtration, we found that the virus removal factors were similar to that of virus infectivity assay. It takes about a day to complete entire assay processes, thus, the TaqMan PCR assay is at least 10-fold faster than the infectivity assay. Therefore, we concluded that this fast, specific, sensitive, and robust assay could replace the infectivity assay for virus clearance evaluation.

Animals↗

PubMed2001

[Detecting human papillomavirus type 6/11 with fluorescence quantitative PCR].

BACKGROUND: To study the human paillomavirus type 6/11 (HPV6/11) infectious status of patients with vaginitis. METHODS: Fluorescence quantitative PCR (FQ-PCR) was used to measue HPV 6/11 DNA in 129 secretion samples. RESULTS: 47 samples were positive with a positive rate of 36.43%; 39 of 47 samples had over 105 copy/ml of the virus. CONCLUSIONS: Middle and old aged patients do not go to hospital until their virus infectious state becomes serious. FQ-PCR is suitable for clinical application because of its high sensitivity, rapidity and accuracy.

Adult↗

PubMed2006

Validation of the 2-DeltaDeltaCt calculation as an alternate method of data analysis for quantitative PCR of BCR-ABL P210 transcripts.

Chronic myelogenous leukemia (CML) is a clonal myeloproliferative disorder that is characterized by the presence of a reciprocal translocation between chromosomes 9 and 22 and results in the formation of the Philadelphia (Ph1) chromosome and is present in most of CML patients. The Ph1 chromosome forms a chimeric gene that encodes an abnormal P210 mRNA transcript in most CML patients. Surveillance for minimal residual disease by detection of BCR/ABL transcripts is currently done mostly by quantitative real-time reverse transcriptase polymerase chain reaction (RT-PCR). Quantitation of BCR/ABL transcripts can monitor tumor load and the outcome of therapy. Absolute quantification determines the input copy number of the transcript of interest, usually by plotting the amount of PCR product onto a standard curve based on serial dilutions of the same product cloned in plasmids. Relative quantification describes the change in expression of the target gene in the patient sample relative to that of a control transcript by using the 2-DeltaDeltaCt calculation. The results of real-time RT-PCR for BCR/ABL transcripts are often analyzed by using plasmid DNA standard curves. In the present study, 79 BCR/ABL transcript-positive samples from CML patients who were being monitored for minimal residual disease by real-time quantitative RT-PCR were studied to determine whether the 2-DeltaDeltaCt approach was equivalent to the plasmid standard curve method. BCR/ABL P210 transcripts were quantitated using both the plasmid standard curve method and the 2-DeltaDeltaCt calculation. The comparison of both methods revealed a highly significant and linear correlation between the plasmid standard curve method and the 2-DeltaDeltaCt calculation (R2=0.98, P<0.0001). Furthermore, there was a reduction of preparation time, contamination risk, and reagent usage. The 2-DeltaDeltaCt calculation is a convenient alternative method to derive accurate quantitative information from real time PCR assays.

Data Interpretation, Statistical↗

PubMed2005

Real-time quantitative PCR for the design of lentiviral vector analytical assays.

From the recent and emerging concerns for approving lentiviral vector-mediated gene transfer in human clinical applications, several analytical methods have been applied in preclinical models to address the lentiviral vector load in batches, cells or tissues. This review points out the oldest generation methods (blots, RT activity, standard PCR) as well as a full description of the newest real-time quantitative PCR (qPCR) applications. Combinations of primer and probe sequences, which have worked in the lentiviral amplification context, have been included in the effort to dress an exhaustive list. Also, great variations have been observed from interlaboratory results, we have tempted to compare between them the different analytical methods that have been used to consider (i) the titration of lentiviral vector batches, (ii) the absence of the susceptible emerging replicative lentiviruses or (iii) the lentiviral vector biodistribution in the organism.

Animals↗

PubMed2002

Quantitative assessment of WT1 expression by real time quantitative PCR may be a useful tool for monitoring minimal residual disease in acute leukemia patients.

In order to verify if quantitative assessment of the WT1 transcript amount by the real time quantitative PCR (RQ-PCR) can be used as a marker for minimal residual disease detection, the WT1 transcript amount was determined in BM and PB samples of patients with myeloid and lymphoid acute leukemia, in normal controls, in regenerating bone marrow samples and in purified CD34-positive cells from normal subjects. In 10 patients bearing a fusion gene transcript suitable for minimal residual disease quantitative assessment, we performed a simultaneous analysis of the WT1 and of the fusion-gene transcript at sequential time intervals during follow-up. Sequential WT1 analysis was also performed in five AML patients lacking additional molecular markers. The data obtained show that normal and regenerating BM samples and purified CD34-positive cells consistently express minimal amounts of WT1 transcript and that this is extremely low and frequently undetectable in normal PB. By contrast, high levels of WT1 expression are present in the BM and PB samples of all acute leukemia (AL) cases at diagnosis. The WT1 levels during follow-up were found to follow the pattern of the other molecular markers (fusion gene transcripts) used for MRD monitoring and increased WT1expression in the BM and/or PB during follow-up of AL patients was always found to be predictive of an impending hematological relapse.

Base Sequence↗

PubMed2005

Sequence variation in primer targets affects the accuracy of viral quantitative PCR.

This study was designed to assess the effect of sequence mismatches between primers and their targets on viral quantitative PCR. Numerous primers were constructed incorporating various mismatches with a target sequence on the BKV T antigen gene. When using these primers in standard Taqman two-step cycling conditions, as few as two mismatches in a single primer increased cycle threshold values and significantly influenced the calculation of viral load.

BK Virus↗

PubMed2001

Combination of competitive quantitative PCR and constant-denaturant capillary electrophoresis for high-resolution detection and enumeration of microbial cells.

A novel quantitative PCR (QPCR) approach, which combines competitive PCR with constant-denaturant capillary electrophoresis (CDCE), was adapted for enumerating microbial cells in environmental samples using the marine nanoflagellate Cafeteria roenbergensis as a model organism. Competitive PCR has been used successfully for quantification of DNA in environmental samples. However, this technique is labor intensive, and its accuracy is dependent on an internal competitor, which must possess the same amplification efficiency as the target yet can be easily discriminated from the target DNA. The use of CDCE circumvented these problems, as its high resolution permitted the use of an internal competitor which differed from the target DNA fragment by a single base and thus ensured that both sequences could be amplified with equal efficiency. The sensitivity of CDCE also enabled specific and precise detection of sequences over a broad range of concentrations. The combined competitive QPCR and CDCE approach accurately enumerated C. roenbergensis cells in eutrophic, coastal seawater at abundances ranging from approximately 10 to 10(4) cells x ml(-1). The QPCR cell estimates were confirmed by fluorescent in situ hybridization counts, but estimates of samples with <50 cells x ml(-1) by QPCR were less variable. This novel approach extends the usefulness of competitive QPCR by demonstrating its ability to reliably enumerate microorganisms at a range of environmentally relevant cell concentrations in complex aquatic samples.

Animals↗

PubMed2004

[Quantitative PCR in the diagnosis of Leishmania].

Polymerase chain reaction (PCR) is a sensitive and rapid method for the diagnosis of canine Leishmania infection and can be performed on a variety of biological samples, including peripheral blood, lymph node, bone marrow and skin. Standard PCR requires electrophoretic analysis of the amplification products and is usually not suitable for quantification of the template DNA (unless competitor-based or other methods are developed), being of reduced usefulness when accurate monitoring of target DNA is required. Quantitative real-time PCR allows the continuous monitoring of the accumulation of PCR products during the amplification reaction. This allows the identification of the cycle of near-logarithmic PCR product generation (threshold cycle) and, by inference, the relative quantification of the template DNA present at the start of the reaction. Since the amplification product are monitored in "real-time" as they form cycle-by-cycle, no post-amplification handling is required. The absolute quantification is performed according either to an internal standard co-amplified with the sample DNA, or to an external standard curve obtained by parallel amplification of serial known concentrations of a reference DNA sequence. From the quantification of the template DNA, an estimation of the relative load of parasites in the different samples can be obtained. The advantages compared to standard and semi-quantitative PCR techniques are reduction of the assay's time and contamination risks, and improved sensitivity. As for standard PCR, the minimal components of the quantitative PCR reaction mixture are the DNA target of the amplification, an oligonucleotide primer pair flanking the target sequence, a suitable DNA polymerase, deoxynucleotides, buffer and salts. Different technologies have been set up for the monitoring of amplification products, generally based on the use of fluorescent probes. For instance, SYBR Green technology is a non-specific detection system based on a fluorescent dsDNA intercalator and it is applicable to all potential targets. TaqMan technology is more specific since performs the direct assessment of the amount of amplified DNA using a fluorescent probe specific for the target sequence flanked by the primer pair. This probe is an oligonucleotide labelled with a reporter dye (fluorescent) and a quencher (which absorbs the fluorescent signal generated by the reporter). The thermic protocol of amplification allows the binding of the fluorescent probe to the target sequence before the binding of the primers and the starting of the polymerization by Taq polymerase. During polymerization, 5'-3' exonuclease activity of Taq polymerase digests the probe and in this way the reporter dye is released from the probe and a fluorescent signal is detected. The intensity of the signal accumulates at the end of each cycle and is related to the amount of the amplification product. In recent years, quantitative PCR methods based either on SYBR Green or TaqMan technology have been set up for the quantification of Leishmania in mouse liver, mouse skin and human peripheral blood, targeting either single-copy chromosomal or multi-copy minicircle sequences with high sensitivity and reproducibility. In particular, real-time PCR seems to be a reliable, rapid and noninvasive method for the diagnosis and follow up of visceral leishmaniasis in humans. At present, the application of real-time PCR for research and clinical diagnosis of Leishmania infection in dogs is still foreseable. As for standard PCR, the high sensitivity of real-time PCR could allow the use of blood sampling that is less invasive and easily performed for monitoring the status of the dogs. The development of a real-time PCR assay for Leishmania infantum infection in dogs could support the standard and optimized serological and PCR methods currenly in use for the diagnosis and follow-up of canine leishmaniasis, and perhaps prediction of recurrences associated with tissue loads of residual pathogens after treatment. At this regard, a TaqMan Real Time PCR method developed for the quantification of Leishmania infantum minicircle DNA in peripheral blood of naturally infected dogs sampled before and at different time points after the beginning of a standard antileishmanial therapy will be illustrated.

Animals↗

PubMed2004

Quantitative PCR with 16S rRNA-gene-targeted species-specific primers for analysis of human intestinal bifidobacteria.

A highly sensitive quantitative PCR detection method has been developed and applied to the distribution analysis of human intestinal bifidobacteria by combining real-time PCR with Bifidobacterium genus- and species-specific primers. Real-time PCR detection of serially diluted DNA extracted from cultured bifidobacteria was linear for cell counts ranging from 10(6) to 10 cells per PCR assay. It was also found that the method was applicable to the detection of Bifidobacterium in feces when it was present at concentrations of >10(6) cells per g of feces. Concerning the distribution of Bifidobacterium species in intestinal flora, the Bifidobacterium adolescentis group, the Bifidobacterium catenulatum group, and Bifidobacterium longum were found to be the three predominant species by examination of DNA extracted from the feces of 46 healthy adults. We also examined changes in the population and composition of Bifidobacterium species in human intestinal flora of six healthy adults over an 8-month period. The results showed that the composition of bifidobacterial flora was basically stable throughout the test period.

Adult↗

PubMed2004

SYBR green-based real-time quantitative PCR assay for detection of West Nile Virus circumvents false-negative results due to strain variability.

Real-time quantitative PCR is used routinely for the high-throughput diagnosis of viral pathogens, such as West Nile virus (WNV). Rapidly evolving RNA viruses present a challenge for diagnosis because they accumulate mutations that may render them undetectable. To explore the effect of sequence variations on assay performance, we generated every possible single point mutation within the target region of the widely used TaqMan assay for WNV and found that the TaqMan assay failed to detect 47% of possible single nucleotide variations in the probe-binding site and was unable to detect any targets with more than two mutations. In response, we developed and validated a less expensive assay with the intercalating dye SYBR green. The SYBR green-based assay was as sensitive as the TaqMan assay for WNV. Importantly, it detected 100% of possible WNV target region variants. The assay developed here adds an additional layer of protection to guard against false-negative results that result from natural variations or drug-directed selection and provides a rapid means to identify such variants for subsequent detailed analysis.

Animals↗

PubMed1992

Analysis of quantitative PCR for the diagnosis of deletion and duplication carriers in the dystrophin gene.

A direct, non-radioactive method of quantitative PCR amplification has been investigated for the diagnosis of deletion and duplication carriers in the dystrophin gene. The simultaneous amplification of two loci, or several loci using multiplex PCR, allows for the direct comparison of relative amounts of products from normal homozygous loci and potentially heterozygous deleted/duplicated loci. Sufficient cycles of PCR are performed to enable visual analysis or densitometric quantification of products on ethidium bromide stained gels. The method has been verified in blind trials performed on known genotypes and by showing that under the conditions used the assay remains within the exponential phase of amplification.

Chromosome Deletion↗

PubMed2026

Development and epidemiological investigation of a TaqMan-based multiplex real-time quantitative PCR assay for simultaneous detection of five bovine viruses (BVDV, AKAV, BNoV, BEV, and BCoV).

INRODUCTION: Infectious diseases caused by bovine viral diarrhea virus (BVDV), Akabane virus (AKAV), bovine norovirus (BNoV), bovine enterovirus (BEV), and bovine coronavirus (BCoV) significantly threaten the cattle industry, resulting in substantial economic losses. These pathogens often present similar clinical signs, such as diarrhea, vomiting, and reproductive disorders in pregnant cattle, and frequent covert or mixed infections further complicate accurate diagnosis. Therefore, rapid, sensitive, and field‑deployable diagnostic methods are essential for effective disease surveillance and control in the cattle industry. METHODS: In this study, we report for the first time the establishment of a TaqMan‑based real‑time quantitative PCR (qPCR) assay that enables simultaneous detection of these five bovine viruses. Multiple sequence alignment of conserved genomic regions was performed, and virus‑specific primers and probes were designed and optimized using Beacon Designer 7 software. Subsequently, a TaqMan‑based multiplex real‑time qPCR assay was established for simultaneous detection of BVDV, AKAV, BNoV, BEV, and BCoV. The established detection method was applied to 200 clinical samples collected from 10 farms in multiple regions of Jilin Province. RESULTS: The results showed that the detection rates for BVDV, AKAV, BNoV, BEV, and BCoV were 33.50%, 0.50%, 4.50%, 7.50%, and 12.00%, respectively. Mixed infections were detected in 9 samples co‑infected with two of the five pathogens, with an overall mixed infection rate of 4.50%. Compared with conventional PCR, coincidence rates were 100% for BVDV, AKAV, BNoV, BEV, and BCoV. DISCUSSION: These findings indicate that the TaqMan multiplex real‑time qPCR assay developed here demonstrates favorable specificity, sensitivity, and reproducibility. This assay enables efficient detection and surveillance of bovine viruses, offering a reliable technical tool for the diagnosis and control of corresponding viral diseases in cattle.

Akabane virus (AKAV)↗

PubMed2005

Use of green fluorescent protein-expressing Aspergillus fumigatus conidia to validate quantitative PCR analysis of air samples collected on filters.

This study used green fluorescent protein (GFP)-expressing Aspergillus fumigatus conidia to compare quantitative PCR (qPCR) enumeration with direct epifluorescent microscopic filter counts of conidia collected on filters in a test chamber. In separate experiments this study initially compared white versus fluorescent light microscopy for counting A. fumigatus conidia, then compared fluorescent microscopy counting of corresponding filter halves, and finally compared qPCR enumeration to counting by fluorescent light microscopy. The use of GFP-expressing conidia with epifluorescent microscopy yielded significantly higher conidia counts (p = 0.026, n = 41, mean of 4.1 conidia per counting field) and 40% faster counting times when compared to conventional counting using white light microscopy. GFP-expressing conidia were aerosolized in a test chamber and collected onto filters. Filters were divided in half and GFP-expressing conidia enumerated. There was no significant difference in the average conidia count per field between corresponding filter halves (p = 0.3, n = 9 filters, mean of 7.8 conidia per counting field). Thus, one filter half could be counted optically and would provide a reliable estimate of filter loading of the corresponding half, which could then be analyzed by qPCR. Filters (n = 38) loaded with GFP conidia in the aerosol chamber were divided in half and analyzed by either fluorescent microscopy or qPCR. The estimated filter loadings ranged from 15-30,000 conidia per filter. There was a linear relationship with a nearly 1:1 ratio between qPCR and direct microscopic estimates of filter loading (y = 1.06x + 404; R(2) = 0.91) showing that the outlined qPCR analysis method is in agreement with an external reference method and is reliable for enumerating A. fumigatus conidia collected on filters. The comparative data derived using GFP-expressing conidia confirmed that qPCR provides sensitive and accurate quantification of DNA from airborne conidia collected on filters.

Air Pollution, Indoor↗