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PubMed2003

Characterization of platelet-specific mRNA by real-time PCR after laser-assisted microdissection.

Circulating anucleate platelets contain minute amounts of residual megakaryocytic-derived mRNA. To study cell type-specific gene expression in platelets, an accurate and sensitive method to detect and quantify platelet mRNA that excludes contamination with leukocyte RNA is mandatory. Applying laser-assisted microdissection and manipulation (LMM) we could isolate platelets from hemalaun-stained cytospins under permanent visual control and after laser-photolysis of nucleated blood cells. For mRNA quantification, the platelet-specific mRNAs were subsequently measured by real time RT-PCR. High-copy beta3 integrin and low-copy a alpha2 integrin as well as tissue factor (TF) transcripts were analyzed in LMM-harvested platelets. In 91.2% (83/91) beta3 integrin was detectable with a mean threshold cycle (CT) value of 32.5+/-3.2 (< or =50,000 cells). The low-copy a 2 integrin mRNA was positive in 84.4% (38/45) with CT mean value of 36.9+/-1.3, indicating that the relative expression of alpha2 integrin mRNA in platelets was about 130 times lower than beta3. The TF transcript was undetectable in all samples. Comparing platelet mRNA from LMM isolation to that from limiting dilution series resulted in a high accordance for beta3 integrin transcript in both, recovery (91.2% vs. 95.2%) and CT value (32.5 vs. 32.8). These results demonstrate that the combination of LMM and real-time RT-PCR is a valuable tool for precise, platelet-specific mRNA analysis without contamination of other cells.

Blood Platelets↗
PubMed2005

Choice of endogenous control for gene expression in nonsmall cell lung cancer.

This study attempts to identify a suitable endogenous control gene for real-time RT-PCR in nonsmall cell lung cancer (NSCLC) tissues. Expression of seven common endogenous control genes (glyceraldehyde-3-phosphate dehydrogenase (GAPDH), v-abl Abelson murine leukaemia viral oncogene homologue 1, beta-2-microglobulin, hypoxanthin phosphoribosyltransferase 1 (HPRT1), phosphoglycerate kinase 1, peptidylprolyl isomerase A, and ribosomal protein, large, P0) in 18 heterogenous NSCLC tumour specimens, 10 normal lung tissues and six NSCLC cell lines were analysed by quantitative RT-PCR. The variances and correlation coefficients of cycle threshold (Ct) value of each control gene in three tissue groups and subgroups were compared. The difference and correlation coefficients between the Ct value for each control gene and the mean Ct value of the remaining control genes were calculated. The GAPDH gene transcript showed the least variance and linear regression analysis demonstrated that GAPDH and HPRT had the strongest correlation in pooled tumour and normal lung tissues. Furthermore, GAPDH expression value showed stringent correlation and had the lowest difference with the mean expression value of the remaining endogenous control genes. Among the seven common endogenous control genes, glyceraldehyde-3-phosphate dehydrogenase is the most suitable for quantitative RT-PCR reaction in nonsmall cell lung cancer tissue samples.

Adult↗
PubMed2005

Constitutive expression of Atlantic salmon Mx1 protein in CHSE-214 cells confers resistance to infectious salmon anaemia virus.

Infectious salmon anaemia (ISA) is a highly fatal viral disease affecting marine-farmed Atlantic salmon which is caused by ISA virus (ISAV), a fish orthomyxovirus that has recently been assigned to the new genus Isavirus within the family Orthomyxoviridae. Mx proteins are among the interferon (IFN)-induced proteins responsible for the development of an antiviral state in vertebrate cells. We used real-time reverse transcription-polymerase chain reaction (RT-PCR) and Chinook salmon embryo (CHSE-214) cells constitutively expressing Atlantic salmon Mx1 protein (ASMx1) to examine the antiviral properties of ASMx1 against two ISAV strains, NBISA01 and HKS-36, having phenotypically different growth properties (cytopathic vs non-cytopathic) in the CHSE-214 cell line. We present evidence that ISAV is sensitive to ASMx1. CHSE-214 cells constitutively expressing ASMx1 showed increased resistance to infection with the cytopathic ISAV strain NBISA01, manifested as delayed development of cytopathic effects (CPE) and significant reduction in the severity of CPE, as well as a 10-fold reduction in virus yield. However, by real-time RT-PCR we observed no significant difference in the mean threshold cycle (Ct) values of ISAV RNA levels, suggesting that the ASMx1 activity on ISAV occurs at the post-transcription steps of virus replication, possibly in the cytoplasm.

Animals↗
PubMed2005

In vitro comparison of RNA preparation methods for detection of feline calicivirus in urine of cats by use of a reverse transcriptase-polymerase chain reaction assay.

OBJECTIVE: To compare 5 methods of preparation of RNA from feline urine samples for use in a feline calicivirus (FCV), p30 gene-based, real-time reverse-transcriptase polymerase chain reaction (RT-PCR) assay. SAMPLE POPULATION: Urine and blood samples from 6 specific-pathogen-free cats. PROCEDURES: Aliquots of each urine sample (unmodified, centrifuged, or mixed with whole or hemolyzed blood) were spiked with FCV and serially diluted in urine. Serial dilutions of FCV in tissue culture medium were used as positive controls. Viral RNA was prepared via dilution and thermal inactivation (DT method), polyethylene glycol precipitation (PEG method), isolation with oligo(dT)25-coated magnetic beads (dTMB method), or extraction by use of 2 silica gel-based columns (RN or QA method). Lower detection limits and mean RT-PCR threshold cycle (Ct) values associated with each RNA preparation method and sample type were compared. RESULTS: Because DT-prepared samples yielded negative results via RT-PCR assay, this method was not evaluated. Lower detection limits (TCID50/sample) for the assay in urine were 1950, 104, 11, and 7 for PEG-, dTMB-, RN-, and QA-prepared samples, respectively. For RN and QA preparations, Ct values were similar and significantly lower than those for dTMB and PEG preparations. Overall, urine modifications did not affect FCV RNA detection in dTMB-, QA-, and RN-prepared samples. CONCLUSIONS AND CLINICAL RELEVANCE: Of the methods evaluated, the RN and QA methods of RNA preparation were most appropriate for the FCV RT-PCR assay. An RT-PCR assay optimized for detection of FCV in feline urine may aid investigations of FCV-induced urinary tract diseases in cats.

Animals↗
PubMed2005

The heritability of the expression of two stress-regulated gene fragments in pigs.

Pigs reared in commercial production units sometimes encounter stressors that significantly decrease growth performance. It is hypothesized that response to stress challenges could potentially be used as selection criteria. This study aimed to investigate, in a commercial setting, the heritability of two target genes previously shown to be induced in response to stress, and related to growth performance, in an experimental situation. Blood samples (n = 2,392) were collected from three separate breeding lines of pedigreed and performance-tested boars between 24 to 25 wk of age. The expression levels of a novel fragment, '29a,' and the calcitonin receptor gene (CTR) were quantified using quantitative real-time PCR (qRT-PCR) on a subset (n = 709) of the blood samples. Gene expression levels were corrected for the efficiency of PCR reactions and also computed directly from threshold cycle (Ct) values. Resulting data showed a skewed nonnormal distribution of expression levels for the target genes relative to the endogenous control, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and were highly variable. Analyses were subsequently performed using untransformed and log-transformed data, with outliers identified and deleted in edited data sets. Regardless of the transformation or editing procedures for outliers applied, there was negligible genetic variation for the expression of target genes relative to GAPDH. In contrast, repeatabilities of replicate samples were generally high (between 0.54 and 0.67). Absolute expression levels for GAPDH and 29a were lowly heritable (h2 of about 0.04), although estimates did not exceed their SE. Subsetting the data according to whether the target gene had a higher or lower level of expression than GAPDH was then performed using the relevant Ct values. In the subset where the target gene was more highly expressed than GAPDH, a moderate estimate of heritability (0.18 +/- 0.10) for the log-transformed absolute expression level of 29a was obtained, whereas the estimate for its expression relative to GAPDH was lower (0.09 +/- 0.07). Estimates of heritability did not increase in the subset of low expression data. The limitations of using gene expression measures as potential selection criteria in commercial situations are discussed.

Animals↗
PubMed2004

Quantitative real-time RT-PCR demonstrates that handling stress can lead to rapid increases of gill-associated virus (GAV) infection levels in Penaeus monodon.

Gill-associated virus (GAV) of the black tiger prawn Penaeus monodon has been implicated as a cause of periodic production losses in Australia since 1996. We report here the development of a real-time quantitative RT-PCR (qRT-PCR) for GAV. A dilution series of in vitro transcribed RNA was used to determine the sensitivity limit of the qRT-PCR and as a standard for GAV quantification. A linear relationship between cycle threshold (Ct) values and input RNA was obtained over a wide concentration range between 4.86 x 10(9) and 0.5 template copies per reaction, the latter being the test detection limit. The qRT-PCR was used to follow the progression of GAV levels in a group of 15 adult male P. monodon with chronic GAV infections that were super-infected by intramuscular injection of an inoculum containing high levels of GAV. By Day 9 post-injection, cumulative mortalities reached 100% (15/15) in the GAV-injected prawns and 40% (2/5) in placebo-injected prawns. Spermatophores were collected at the beginning, and together with other tissues, at the end of the trial. Prawns were also bled at regular intervals to collect circulating haemocytes. The qRT-PCR revealed that GAV loads increased significantly in haemocytes collected from both the control and super-infected prawns (p = 0.010). This increase was significantly higher in the super-infected prawns (p = 0.047). The rapid increase in GAV levels in super-infected P. monodon was expected. However, the increase in the control prawns was not, and indicates that repetitive bleeding and handling stress can stimulate GAV proliferation in chronically infected P. monodon.

Animals↗
PubMed2006

Development and evaluation of a one-step real-time reverse transcription polymerase chain reaction assay for the detection of salmonid alphaviruses in serum and tissues.

We designed 4 primer pairs to amplify conserved regions of the E1 or nsP4 genes of salmonid alphavirus (SAV) and evaluated their performance in optimized 1-step SYBR green real-time RT-PCR (RRT-PCR) assays. A single primer pair, amplifying a 227 bp segment of E1 was then chosen for further study. This RRT-PCR was shown to be highly repeatable and reproducible over a wide range of RNA dilutions, with a linear relationship between cycle threshold (Ct) value and RNA concentration over a 10(7) dilution range. The limit of detection was calculated to be < or = 1.5 TCID50 ml(-1). When applied to sera previously screened by virus isolation for SAV viraemia, the RRT-PCR correctly identified all 13 culture-positive samples, as well as finding an additional 28 sera positive. Relative semi-quantitation of sera showed a very highly significant relationship between copy number and TCID50 (p < 0.001, R2 = 0.9563). Following experimental infection of salmon, heart samples were consistently positive until 21 d post infection (dpi), with (weak) positive signals still detectable in 50% of fish 70 dpi.

Alphavirus↗
PubMed2000

Detection of MUC1-expressing mammary carcinoma cells in the peripheral blood of breast cancer patients by real-time polymerase chain reaction.

We have prospectively analyzed blood samples of 122 patients with breast disease for the presence of circulating expressing MUC1 cells before and after treatment. Among them, 28 patients had histologically confirmed benign breast disease (group 1), 34 patients had operable breast cancer (group 2), and 60 patients had advanced breast cancer (group 3). Circulating epithelial cells were isolated with BerEP4-coated immunomagnetic beads. Total RNA was extracted and reverse transcribed before analysis by real-time PCR of a MUC1-specific cDNA sequence. The sensitivity of the reverse transcription-PCR tested with blood spiked with MCF7 cells was one cell in 5 ml of blood. The immunomagnetic separation step was mandatory to obtain the maximum specificity. Control samples from healthy donors never displayed cycle threshold (Ct) values for MUC1 lower than 38. Circulating cells (Ct, <38) were detected in 3 of 28 (11%) cases in group 1, in 8 of 34 (24%) cases in group 2, and in 27 of 60 cases (45%) in group 3. A semiquantitative estimate of blood-borne cells could be derived from the Ct value when below 32 (the lowest was 28) or by the number of positive aliquots of the same blood sample. Thus, immunomagnetic separation, followed by MUC1-specific RT-PCR, allows the semiquantitative detection of circulating mammary cells. A significant correlation between the presence of MUC1-positive cells and the group of breast tumors was observed. The clinical significance of blood-borne cells in breast cancer, especially at the operable stage, may be investigated by following these patients.

Adult↗
PubMed2006

Age-related changes in relative expression of real-time PCR housekeeping genes in human skeletal muscle.

The purpose of this investigation was to examine the expression of three commonly used housekeeping genes -- glyceraldehyde-3-phosphate dehydrogenase (GAPDH), beta(2)-microglobulin (beta(2)M), and RNA polymerase 2a (polR2a) -- in elderly (E) compared to young (Y) subjects. Nine young subjects (22.7 +/- 3.4 yrs) and 11 elderly subjects (73.0 +/- 9.5 yrs) underwent a percutaneous skeletal muscle biopsy of the vastus lateralis. Equal concentrations of isolated mRNA from these samples were used to perform real-time polymerase chain reaction with primer/probe combinations specific to each gene of interest. The expression of GAPDH, beta(2)M, and polR2a was obtained as the value of cycle threshold (C(T)). An independent t-test with a level of significance at p < or = 0.05 was used to determine differences between groups. There was no difference in average C(T) of GAPDH between groups (p=0.869) (Y = 16.92 +/- 2.25 vs. E = 17.08 +/- 2.09) and polR2a (p = 0.089) (Y = 28.00 +/- 0.89 vs. E = 26.73 +/- 1.91). However, there was a significant difference (p < or = 0.05) in the average C(T) of beta(2)M (Y =21.79 +/- 0.44 vs. E = 21.05 +/- 0.51). The results indicate that special consideration needs to be made when selecting housekeeping genes for comparisons in real-time reverse-transcriptase polymerase chain reaction, depending upon the age of the populations of interest.

Adult↗
PubMed2004

Quantitative real-time polymerase chain reaction based on single copy gene sequence for detection of periodontal pathogens.

OBJECTIVE: To develop a method for quantification of Actinobacillus actinomycetemcomitans (Aa), Porphyromonas gingivalis (Pg) and Tannerella forsythensis (Tf) from subgingival plaque samples based on TaqMan real-time polymerase chain reaction (PCR) technology. MATERIAL AND METHODS: Bacterial cells from these species were obtained after culturing reference strains and were counted microscopically. Cellular suspensions in Tris-EDTA buffer were used for DNA extraction after boiling for 20 min. Primers for PCR were selected from sequences of the LktC (Aa), Arg-gingipain (Pg) and BspA antigen (Tf) genes in order to yield amplicons below 100 bp. TaqMan-based real-time PCR was adjusted to quantify each species separately. Cycle threshold (C(T)) values were calculated for each species according to the initial number of copies. A reliability analysis was carried out using intra-class correlation coefficients (ICCs) with a two-way random effects model. RESULTS: A high sensitivity and specificity was obtained for the detection of the three bacterial species. The TaqMan real-time PCR technology yielded a good repeatability in the obtained cycle threshold (C(T)) values for each initial number of copies, demonstrating coefficients of variation below 5% for each bacteria. The reproducibility of the technique was also demonstrated by the high ICCs (>0.98; p<0.00001) obtained for each bacteria with and without the addition of subgingival plaque. CONCLUSION: A novel diagnostic method based on TaqMan real-time PCR was developed for the quantification of Aa, Pg and Tf. It has demonstrated good sensitivity and repeatability on pure cultures. Its diagnostic utility should be demonstrated in subgingival plaque samples.

Aggregatibacter actinomycetemcomitans↗
PubMed2002

Influence of cycling cadence on subsequent running performance in triathletes.

PURPOSE: The purpose of this study was to investigate the influence of different cycling cadences on metabolic and kinematic parameters during subsequent running. METHODS: Eight triathletes performed two incremental tests (running and cycling) to determine maximal oxygen uptake (VO2max) and ventilatory threshold (VT) values, a cycling test to assess the energetically optimal cadence (EOC), three cycle-run succession sessions (C-R, 30-min cycle + 15-min run), and one 45-min isolated run (IR). EOC, C-R, and IR sessions were realized at an intensity corresponding to VT + 5%. During the cycling bouts of C-R sessions, subjects had to maintain one of the three pedaling cadences corresponding to the EOC (72.5 +/- 4.6 rpm), the freely chosen cadence (FCC; 81.2 +/- 7.2 rpm), and the theoretical mechanical optimal cadence (MOC, 90 rpm; Neptune and Hull, 1999). RESULTS: Oxygen uptake (VO2) increased during the 30-min cycling only at MOC (+12.0%) and FCC (+10.4%). During the running periods of C-R sessions, VO2, minute ventilation, and stride-rate values were significantly higher than during the IR session (respectively, +11.7%, +15.7%, and +7.2%). Furthermore, a significant effect of cycling cadence was found on VO2 variability during the 15-min subsequent run only for MOC (+4.1%) and FCC (+3.6%). CONCLUSION: The highest cycling cadences (MOC, FCC) contribute to an increase in energy cost during cycling and the appearance of a VO2 slow component during subsequent running, whereas cycling at EOC leads to a stability in energy cost of locomotion with exercise duration. Several hypotheses are proposed to explain these results such as changes in fiber recruitment or hemodynamic modifications during prolonged exercise.

Adult↗
PubMed2003

Effects of creatine supplementation on housekeeping genes in human skeletal muscle using real-time RT-PCR.

The present study examined the validity and reliability of measuring the expression of various genes in human skeletal muscle using quantitative real-time RT-PCR on a GeneAmp 5700 sequence detection system with SYBR Green 1 chemistry. In addition, the validity of using some of these genes as endogenous controls (i.e., housekeeping genes) when human skeletal muscle was exposed to elevated total creatine levels and exercise was also examined. For all except 28S, linear relationships between the logarithm of the starting RNA concentrations and the cycle threshold (C(T)) values were established for beta-actin, beta2-microglobulin (beta2M), cyclophilin (CYC), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). We found a linear response between C(T) values and the logarithm of a given amount of starting cDNA for all the genes tested. The overall intra-assay coefficient of variance for these genes was 1.3% and 21% for raw C(T) values and the linear value of 2(-C(T)), respectively. Interassay variability was 2.3% for raw C(T) values and 34% for the linear value of 2(-C(T)). We also examined the expression of various housekeeping genes in human skeletal muscle at days 0, 1, and 5 following oral supplementation with either creatine or a placebo employing a double-blind crossover study design. Treatments were separated by a 5-wk washout period. Immediately following each muscle sampling, subjects performed two 30-s all-out bouts on a cycle ergometer. Creatine supplementation increased (P < 0.05) muscle total creatine content above placebo levels; however, there were no changes (P > 0.05) in C(T) values across the supplementation periods for any of the genes. Nevertheless, 95% confidence intervals showed that GAPDH was variable, whereas beta-actin, beta2M, and CYC were the least varying genes. Normalization of the data to these housekeeping genes revealed variable behavior for beta2M with more stable expressions for both beta-actin and CYC. We conclude that, using real-time RT-PCR, beta-actin or CYC may be used as housekeeping genes to study gene expression in human muscle in experiments employing short-term creatine supplementation combined with high-intensity exercise.

Actins↗
PubMed1995

Computer simulation of the propagation of contrast medium in a coronary artery during one cardiac cycle.

In some angiographic methods for measurement of mean coronary flow in ml/min, a threshold is applied to 'concentration-distance' curves obtained from a constant rate injection by computing the intravascular contrast medium concentration along the main coronary branches. If the shape of the velocity profile would remain parabolic throughout the cardiac cycle, the correct threshold value would be '50% of the concentration at the injection site'. But, coronary flow being strongly pulsatile, the shape of the velocity profile must be expected to vary appreciably within the cardiac phase. In order to investigate if a single, appropriate threshold value nevertheless exists for a great variety of coronary flow pulses and velocity profiles, the spreading of contrast medium injected continuously in a tube perfused by a time varying flow Q(t) was studied by computer simulation. While the particular time courses of flow and velocity profile appear to be of secondary importance, the ratio 'injection rate to peak coronary flow' has a major impact. If it is equal to or greater than 1, a threshold value of 47% is the best choice. If the ratio is markedly less than 1, no appropriate threshold exists and use of the 47% threshold will result in considerable flow underestimations. This was fully confirmed by measurements of absolute coronary flow performed in patients.

Blood Flow Velocity↗
PubMed2007

Real-time PCR for quantifying Haemonchus contortus eggs and potential limiting factors.

The purpose of this study was to evaluate the practicality of using real-time PCR for quantifying feces-derived trichostrongyle eggs. Haemonchus contortus eggs were used to evaluate fecal contaminants, time after egg embryonation, and the presence of competing and non-competing DNAs as factors that might interfere with generating reproducible results during simplex and multiplex quantitative real-time PCR (QPCR). Real-time PCR results showed linear quantifiable amplification with DNA from five to 75 eggs. However, threshold cycle (C (T)) values obtained by amplification of DNA from egg numbers between 75 and 1,000 did not differ significantly. Inhibitors of QPCR were effectively removed during DNA extraction as exemplified by the absence of any improvement in C (T) values with bovine serum albumin or phytase treatments. Changes from egg embryonation could only be detected during the first 6 h. Noncompetitive DNA did not appear to impact amplification; however, in a multiplex reaction a competing trichostrongyle such as Cooperia oncophora can hinder amplification of H. contortus DNA, when present at tenfold greater amounts. This study demonstrates the usefulness of QPCR for amplification and quantification of trichostrongyle eggs, and identifies potential limitations, which may be addressed through multiplex assays or the addition of a standard: exogenous DNA target.

Animals↗
PubMed2006

Real-time telomeric repeat amplification protocol using the duplex scorpion and two reverse primers system: the high sensitive and accurate method for quantification of telomerase activity.

BACKGROUND: Real-time quantitative TRAP assays for detection of telomerase activity have been recently developed to eliminate complex post-PCR procedures. However, all of them use the conventional TRAP assay that possesses an unpredictable cascade of events in PCR amplification caused by stagger annealing, which may affect the accuracy of quantitation. METHODS: A novel RTQ-TRAP method was developed by combining the duplex scorpion with modified TP-TRAP assay that has high fidelity PCR amplification of the telomerase product (DS/TP-TRAP). The synthesized oligonucleotide that represents telomerase products is used to set up a standard curve. RESULTS: The DS/TP-TRAP method gives the standard curve a dynamic range of 6 orders of magnitude (R(2)=0.9992). It optimizes PCR amplification efficiency and determines telomerase activity in a lower threshold cycle number (Ct value). The method is both accurate and reproducible to measure telomerase activity in human tumor cell lines, and linearity from 1 to 1000 cells could be obtained (R(2)=0.9926). For tumor samples, the results determined by the DS/TP-TRAP assay are comparable to the data obtained with the conventional TRAP method. CONCLUSIONS: The DS/TP-TRAP assay provides a high sensitive and accurate method for real-time quantitative detection of telomerase activity. It is thus a potential robust tool for application in cancer molecular diagnostics.

Base Sequence↗
PubMed2004

Real time PCR for monitoring regulation of host gene expression in herpes simplex virus type 1-infected human diploid cells.

Herpes simplex virus type 1 (HSV-1) induces prominent shifts in the rates of transcription of host cellular genes of relevance for the outcome of the viral infection. The quantitative analysis of transcription may be obscured by virus-induced alterations in the levels of RNA encoded by cellular housekeeping genes that are used commonly for normalisation of real time reverse transcription PCR (RT-qPCR). In the present study, we analysed beta-actin, GAPDH and 18S rRNA for their usefulness in normalisation of RT-qPCR analysis of the transcription of the HSV-1 gamma gB-1 gene and FUT5, a cellular gene induced by viral infection. The transcription of these genes was monitored in a TaqMan-based real time RT-PCR system over a 24h interval of virus infection of human embryonic lung fibroblasts. The levels of gB-1 and FUT5 RNA were normalised via difference in the threshold cycle (deltaC(t)) values relative to each and one of the housekeeping genes or calculated in relation to the number of infected cells without any further normalisation. The levels of RNA encoded by beta-actin or GAPDH were found to vary by several orders of magnitude during HSV-1 infection, introducing large errors in the estimation of the gB-1 and FUT5 RNA levels. In contrast, the variation of C(t) values for 18S rRNA was less than one cycle during 24h period of HSV-1 infection. The FUT5 and gB-1 RNA figures obtained by DeltaC(t) normalisation relative 18S rRNA were identical to those calculated in relation to the number of infected cells. These data recommend 18S rRNA for normalisation in HSV-1-infected human cells but discourage the use of beta-actin and GAPDH RNA for this purpose. By applying these procedures, it was shown that the transcription of FUT5 was increased by 50-fold 5-24h after HSV-1 infection and 200-fold by the inhibition of viral DNA replication in HSV-infected cells.

Actins↗
PubMed2004

Development of a highly sensitive semi-quantitative real-time PCR and molecular beacon probe assay for the detection of respiratory syncytial virus.

Molecular beacons are a novel class of oligonucleotide probe capable of reporting the accumulation of target amplicon during real-time PCR by the emission of a fluorescent signal. A novel assay for the detection and estimation of respiratory syncytial virus (RSV) nucleic acid in clinical specimens based on real-time PCR utilising such a probe was developed. The probe consisted of two short arm sequences and a central loop sequence complementary to a region of the N gene (the target amplicon). The probe was characterised and a semi-quantitative nested real-time PCR using a LightCycler instrument was optimised. Standard curves were generated using cycle threshold (C(t)) values calculated from several assays over a range of logarithmic RSV titres. Linear coefficient correlations were close to one ( r(2) = 0.998) and the detection limit of the optimised assay was reproducibly shown to be 1 x 10 (-4) pfu/ml. The intra-assay coefficient of variation (CV) of C(t) values of the optimal assay was 0.8% and the CV of quantification data was 6.6%. The interassay CV of C(t) values was 2.0% and the quantification CV was 6.7%. The validity of the assay for the detection of RSV in clinical specimens was assessed by analysing ten specimens previously assayed in a different laboratory. Real-time PCR analysis was completely consistent with the results of prior analysis. The rapidity, sensitivity and specificity of the assay should greatly facilitate epidemiological studies, particularly in adults as existing methods perform better on clinical specimens from children.

Humans↗
PubMed2005

Discrimination of lamivudine resistant minor HIV-1 variants by selective real-time PCR.

A selective real-time PCR (SPCR) method was developed and evaluated for discrimination of resistance mutations in minor human immunodeficiency virus type 1 (HIV-1) populations, using the M184 mutation site as a model system due to its high clinical importance and the low genetic barrier to its development. The method enabled detection of minor viral populations down to 0.1%, and the relative proportions of different quasispecies could be easily displayed using cycle threshold (C(t)) values. An excellent concordance was found when the assay was compared with direct sequencing and cloning results. The impact of mismatch between virus and primer/probe sequences was evaluated, showing that 3' end mutations in the selective downstream primers were very disruptive and that 5' end polymorphisms in the probe area were directly fatal, while mutations in the middle or the 3' end of the probe were less disruptive. These effects were compensated by introducing wobble bases to accommodate the mutations. This sensitive and reliable point-mutation assay, analyzing M184I/V and other important mutations, will be fruitful in gaining new scientific knowledge about the kinetics of resistance mutations in minor viral populations of HIV-1 infected patients at failure of antiretroviral therapy.

DNA Primers↗