Fluorogenic Method Research Articles

Fluorescence “Turn-On” chemosensor for the selective detection of beryllium

A new fluorogenic method for selective and sensitive determination of beryllium using 2,6-diphenyl-4-benzo-9-crown-3-pyrane (DBCP) was developed. The proposed fluorescent probe undergoes fluorescent emission intensity enhancement upon binding to beryllium ions in MeOH/H 2O (70:30, v/v) solution. The fluorescence enhancement of DBCP is attributed to a 1:1 complex formation between DBCP and Be 2+ ion, which has been utilized as the basis for selective detection of Be 2+ ion. With the optimum condition described, the fluorescence enhancement at 531 nm was linear to the concentration of beryllium in the range of 1.6 × 10 −8–1.6 × 10 −7 M and a detection limit of 1.5 × 10 −9 M. The fluorescent probe exhibits high selectivity for Be 2+ ion over the other common mono, di- and trivalent cations.

A fluorogenic method for measuring enteropeptidase activity: spectral shift in the emission of GD4K-conjugated 7-amino-4-methylcoumarin

Enteropeptidase is a serine protease secreted by the pancreas and converts inactive trypsinogen to active trypsin. Enteropeptidase cleaves the C-terminal end of the substrate recognition sequence Asp-Asp-Asp-Asp-Lys (D(4)K). The assay for enteropeptidase has utilized GD(4)K-conjugated 2-naphthylamine (GD(4)K-NA) as a fluorogenic probe over the last 30 years. However, no other D(4)K-conjugated fluorogenic substrates of enteropeptidase have been reported. Furthermore, naphthalene is known as carcinogenic to humans. In this study, we used shift in the emission spectrum of GD(4)K-conjugated 7-amino-4-methylcoumarin (GD(4)K-AMC) as a fluorogenic method to measure enteropeptidase activity. The kinetic analysis revealed that enteropeptidase has a K(M) of 0.025 mM and a k(cat) of 65 sec(-1) for GD(4)K-AMC, whereas it has a K(M) of 0.5 to 0.6 mM and a k(cat) of 25 sec(-1) for GD(4)K-NA. The optimum pH of GD(4)K-AMC hydrolysis was pH 8.0. Our data indicate that GD(4)K-AMC is more suitable as a substrate for enteropeptidase than GD(4)K-NA.

Continuous Localized Monitoring of Plasmin Activity Identifies Differential and Regional Effects of the Serine Protease Inhibitor Aprotinin: Relevance to Antifibrinolytic Therapy

Antifibrinolytic therapy, such as the use of the serine protease inhibitor aprotinin, was a mainstay for hemostasis after cardiac surgery. However, aprotinin was empirically dosed, and although the pharmacological target was the inhibition of plasmin activity (PLact), this was never monitored, off-target effects occurred, and led to withdrawn from clinical use. The present study developed a validated fluorogenic microdialysis method to continuously measure PLact and tested the hypothesis that standardized clinical empirical aprotinin dosing would impart differential and regional effects on PLact. Pigs (30 kg) were instrumented with microdialysis probes to continuously measure PLact in myocardial, kidney, and skeletal muscle compartments (deltoid) and then randomized to high-dose aprotinin administration (2 mKIU load/0.5 mKIU/hr infusion; n = 7), low-dose aprotinin administration (1 mKIU load/0.250 mKIU/hr infusion; n = 6). PLact was compared with time-matched vehicle (n = 4), and PLact was also measured in plasma by an in vitro fluorogenic method. Aprotinin suppressed PLact in the myocardium and kidney at both high and low doses, indicative that both doses exceeded a minimal concentration necessary for PLact inhibition. However, differential effects of aprotinin on PLact were observed in the skeletal muscle, indicative of different compartmentalization of aprotinin. Using a large animal model and a continuous method to monitor regional PLact, these unique results demonstrated that an empirical aprotinin dosing protocol causes maximal and rapid suppression in the myocardium and kidney and in turn would likely increase the probability of off-target effects and adverse events. Furthermore, this proof of principle study demonstrated that continuous monitoring of determinants of fibrinolysis might provide a novel approach for managing fibrinolytic therapy.

A simple fluorogenic method for determination of acid ceramidase activity and diagnosis of Farber disease

Acid ceramidase (aCDase) is one of several enzymes responsible for ceramide degradation within mammalian cells. As such, aCDase regulates the intracellular levels of the bioactive lipid ceramide. An inherited deficiency of aCDase activity results in Farber disease (FD), also called lipogranulomatosis, which is characterized by ceramide accumulation in the tissues of patients. Diagnosis of FD is confirmed by demonstration of a deficient aCDase activity and the subsequent storage of ceramide. Existing methods include extremely complex assays, many of them using radiolabeled compounds. Therefore, the aCDase assay and the in vitro enzymatic diagnosis of FD are still performed in only a very limited number of specialized laboratories. Here, the new fluorogenic substrate Rbm14-12 was synthesized and characterized as a new tool to determine aCDase activity. The resulting optimized assay was performed in 96-well plates, and different fibroblast and lymphoid cell lines derived from FD patients and controls were tested to measure aCDase activity. As a result, the activity in cells of FD patients was found to be very low or even null. This new fluorogenic method offers a very easy and rapid way for specific and accurate determination of aCDase activity and, consequently, for diagnosis of FD.

Effective Light-Upon-Extension Real-Time PCR Primer Systems for Rapid Detection of Human Viruses

Background: Real-time polymerase chain reaction (PCR) assays for quantitative detection and typing of viruses are fast, simple, and provide valuable information about the progression of an infection. Identification of human papilloma and polyomaviruses is used to estimate the risk of cancer (papilloma), polyomavirus-associated-nephropathy (BKV), or progressive multifocal leukoencephalopathy (JCV) in individual patients. Thus, non-invasive and specific diagnostics for viral detection should be regarded. Methods: We describe the design, optimization, and application of effective LightUpon Extension (LUX) real-time PCR systems for detection of papillomaviruses, BKV, and JCV. Viral and human controls along with clinical samples were examined during the optimization process. D-LUX designer software and the Opticon 2 Real-Time System (Bio-Rad, Hercules, CA) were used. Results: We confirm the specificity, rapidity, and non-invasiveness of the method for demonstration of ongoing replication. We believe LUX technology is comparable to probe based real-time PCR assays for the detection of viruses, but it has an advantage in terms of cost-effectiveness, convenience, and possibility for a dissociation analysis. Conclusions: The fluorogenic primer method (LUX) is rapid, uses few toxic chemicals, and is useful for the specific and accurate detection of viral DNA among different types of samples.

Group IIA secretory phospholipase A2 (GIIA) mediates apoptotic death during NMDA receptor activation in rat primary cortical neurons

Phospholipases A(2) (PLA(2)) participate in neuronal death signalling pathways because of their ability to release lipid mediators, although the contribution of each isoform and mechanism of neurotoxicity are still elusive. Using a novel fluorogenic method to assess changes in a PLA(2) activity by flow cytometry, here we show that the group IIA secretory phospholipase A(2) isoform (GIIA) was specifically activated in cortical neurons following stimulation of N-methyl-d-aspartate glutamate receptor subtype (NMDAR). For activation, GIIA required Ca(2+) and reactive oxygen/nitrogen species, and inhibition of its activity fully prevented NMDAR-mediated neuronal apoptotic death. Superoxide, nitric oxide or peroxynitrite donors stimulated GIIA activity, which mediated neuronal death. Intriguingly, we also found that GIIA activity induced mitochondrial superoxide production after NMDAR stimulation. These results reveal a novel role for GIIA in excitotoxicity both as target and producer of superoxide in a positive-loop of activation that may contribute to the propagation of neurodegeneration.

A NEW FLUOROGENIC ASSAY FOR MONITORING AND DETERMINING PLANKTONIC AND BIOFILM FORMS OF PSEUDOMONAS AERUGINOSA VIABLE COUNT IN VITRO

ABSTRACT A new method was developed to rapidly monitor the Pseudomonas aeruginosa viable counts using alamar blue (AB). The 96‐well microtiter plates were used to perform the assay. This procedure is based on fluorogenic measurement as a result of reduction of nonfluorescent AB to red fluorescent form by the viable cells of P. aeruginosa. The correlation between conventional plate count and fluorogenic AB method was highly satisfactory for quantification of planktonic (R2 = 0.9487) and biofilm cells of P. aeruginosa (R2 = 0.9296).PRACTICAL APPLICATIONSThe new fluorogenic method can rapidly monitor Pseudomonas aeruginosa counts in vitro with a high correlation with the conventional plating method. The results indicate that fluorogenic method requires much shorter time (2 h) than the conventional plate count (24 h), is a more cost‐effective way, quite amenable to high throughput, and continuous monitoring of P. aeruginosa viability is achievable in the kinetic in vitro models without interference with the cell viability.

A real-time fluorescence method for enzymatic characterization of specialized human DNA polymerases

Specialized DNA polymerases are involved in DNA synthesis during base-excision repair and translesion synthesis across a wide range of chemically modified DNA templates. Notable features of these enzymes include low catalytic efficiency, low processivity and low fidelity. Traditionally, in vitro studies of these enzymes have utilized radiolabeled substrates and gel electrophoretic separation of products. We have developed a simple homogeneous fluorescence-based method to study the enzymology of specialized DNA polymerases in real time. The method is based on fluorescent reporter strand displacement from a tripartite substrate containing a quencher-labeled template strand, an unlabeled primer and a fluorophore-labeled reporter. With this method, we could follow the activity of human DNA polymerases β, η, ι and κ under different reaction conditions, and we investigated incorporation of the aberrant nucleotide, 8-oxodGTP, as well as bypass of an abasic site or 8-oxoG DNA template lesion in different configurations. Lastly, we demonstrate that the method can be used for small molecule inhibitor discovery and characterization in highly miniaturized settings, and we report the first nanomolar inhibitors of Y-family DNA polymerases ι and η. The fluorogenic method presented here should facilitate mechanistic and inhibitor investigations of these polymerases and is also applicable to the study of highly processive replicative polymerases.

Abstract 4336: Interrogating the Human Myocardial Interstitium during Myocardial Arrest and Reperfusion: Dynamic Changes in Cytokines and Protease Activity

Background. Cytokines, such as the interleukins (IL1β, IL2, IL6) and tumor necrosis factor (TNF) can modulate myocardial structure and function with ischemia/reperfusion (I/R) but dynamic assessment of these biological molecules within the human myocardial interstitium with I/R has not been performed, and the inter-relationship to matrix metalloproteinases activity (MMPact) remains unexplored. Accordingly, a fluorogenic microdialysis method was used to simultaneously measure myocardial interstitial cytokine levels and MMPact in patients during and following I/R. Methods . MMPact was measured in patients (n=13) undergoing cardio-pulmonary bypass (CPB) at baseline, during myocardial arrest and CPB (on-CPB), and immediately following reperfusion and separation from CPB (post-CPB) by a validated in-line microdialysis fluorescent detection system. Myocardial interstitial fluid was subjected to cytokine analysis by high sensitivity multiplex suspension array. Results . Interstitial MMPact increased by over 30% post-CPB and was accompanied by a specific change in cytokine profiles (Figure ). The classical pro-inflammatory molecules such as TNF and IL6 were either not detectable or unchanged, whereas IL1β and IL2 which can be proinflammatory, were increased. Conclusions. These unique results demonstrated that a dynamic cytokine signature occurs within the human myocardial interstitium following I/R and is temporally related to heightened MMP activity. Direct interrogation of the human myocardial interstitium may provide a unique insight into critical signaling pathways which may evoke adverse structural and functional events following I/R.

Sensitive determination of total aliphatic amines in water samples by spectrofluorimetry using the new fluorogenic probe 3-(4-fluorobenzoyl)-2-quinolinecarboxaldehyde

A rapid and highly sensitive fluorogenic method has been developed for the determination of total primary aliphatic amines with a novel fluorogenic derivatizing reagent, 3-(4-fluorobenzoyl)-2-quinolinecarboxaldehyde (FBQCA). FBQCA selectively reacts with amines to form a highly fluorescent isoindole in buffer of pH 9.7 and in the presence of potassium cyanide solution. The fluorescence intensity of derivatized products are detected at λex/λem = 480/546 nm. The results of the experiments showed a linear dynamic range of 1 × 10−8 to 6 × 10−6 mol L−1 with a detection limit (S/N = 3) of 6 nmol L−1. The method was successfully used for the direct spectrofluorimetric determination of aliphatic amines in water samples with recoveries of 95–104%.

Ultra-trace level determination of nitrite in human saliva by spectrofluorimetry using 1,3,5,7-tetramethyl-8-(3,4-diaminophenyl)-difluoroboradiaza-s-indacene

A rapid, highly sensitive and selective fluorogenic method for the determination of traces of nitrite is described. It is based on the reaction of weakly fluorescent 1,3,5,7-tetramethyl-8-(3,4-diaminophenyl)-difluoroboradiaza-s-indacence (DAMBO) and nitrite in acidic aqueous solution to give 1,3,5,7-tetramethyl-8-(5-benzotriazolyl)-difluoroboradiaza-s-indacene (DAMBO-T), which is highly fluorescent. The optimum reaction conditions and other analytical parameters are investigated to enhance the sensitivity of the method. The fluorescence enhancement at 507 nm is linearly related to the concentration of nitrite in the range of 6.0 × 10−9–5.0 × 10−7 mol L−1 with a correlation coefficient of R = 0.9995 (n = 10) and a detection limit of 1.0 × 10−10 mol L−1. The R.S.D. is 1.12% (n = 10). The method is applied to the determination of nitrite in human saliva samples with the recoveries of 96. 24–105.30%.

A new rapid fluorogenic method for measuring bacteriocin activity

We describe a novel bacteriocin screening assay based on fluorescence emitted by berberine following its influx into compromised cells. This technique showed agreement with the conventional well-diffusion method, and results can be obtained within one hour. This assay could facilitate the rapid identification of bacteriocinogenic bacterial isolates.

Monocyte IL-10 produced in response to lipopolysaccharide modulates thrombin generation by inhibiting tissue factor expression and release of active tissue factor-bound microparticles

Lipopolysaccharide (LPS)-stimulated monocytes are known to have a procoagulant effect. This property is currently explained by the fact that monocytes, in response to LPS, can express tissue factor (TF) and undergo a process of membrane microvesiculation. Interleukin-10 (IL-10) has been shown to downregulate TF expression and inhibit procoagulant activity (PCA). In order to further characterize the inhibitory effect of IL-10 on LPS-induced PCA, we used the integrated system of analysis of kinetics of thrombin generation in normal plasma (thrombinography). For this, we developed an original method of elutriation allowing to obtain a highly purified monocyte preparation, under endotoxin-free conditions. Thrombin generation was measured using a highly sensitive and specific fluorogenic method which we adapted to inhibit the contact factor pathway. Results show that recombinant human IL-10 decreased the kinetics of thrombin generation in a dose-dependent manner. Furthermore, the inhibition of endogenous IL-10 released by monocytes in response to LPS is associated with an increase in the kinetics of thrombin generation. We demonstrated that this effect was a consequence of the up-regulation of TF expression and TF-bound microparticle release. In conclusion, we report that IL-10 can regulate thrombin generation in conditions close to physiology as allowed by thrombinography, and that endogenous IL-10 regulates TF expression and release of active TF-bound microparticles by a negative feed back loop through IL-10 receptor alpha.

Inter-Laboratory Variation and the Effect of the Type of Fluorimeter on the Results of the Technothrombin® Thrombin Generation Assay.

Thrombin generation is an essential step in the clotting system. Therefore the measurement of thrombin generation is a desirable parameter to elucidate deviations in the clotting system. However till recently the measurement of thrombin generation was a difficult and laborious laboratory method. Nowadays commercial tests are available at the market. We evaluated in a multicenter trial the Technothrombin® TGA assay from Technoclone GmbH Vienna, Austria with a focus on inter-laboratory variability. The Technothrombin® TGA assay is a fluorogenic method. To our knowledge this was the first large-scale inter-laboratory comparison of a thrombin generation test. A set of 8 different samples were distributed to 45 clinical centers in Europe. Thirty-six returned results (80%). Samples were selected on a different level of thrombin generation as indicated by the thrombin concentration at peak height (PTC). The PTC in the samples used varied from 4 nM to 335 nM. The inter-laboratory variability varied from 16% for high PTC (~335 nM) to about 100% for low PTC (~4 - 15 nM).Using a normal citrated pooled plasma as a reference the other samples can be significantly discriminated by all participants (P < 0.03) using an activator with a low phospholipid concentration (0.32 μM) in the test. With a high phospholipid concentration (3.2 μM) two of the samples can not be significantly discriminated from normal pooled plasma.Furthermore, we evaluated the effect of the type of fluorimeter used by the clinical laboratories. The most frequently used fluorimeters in this study were the Biotek FLx 800 (n=11), Thermo Fluoroskan (n=8), Tecan Genios (n=5) and the BMG Fluostar (n=4). Overall there are no significant differences between the measurement of the PTC by the different fluorimeters. However remarkable differences were observed for the inter-laboratory variability between the different fluorimeters. The average coefficient of variation of all samples increased from BMG (19.3% ; range 11.1 - 26.9%) < Biotek (28.8% ; range 15.9 - 59.3%) < Tecan (38.5% ; range 12.6 - 81.5%) < Thermo (49.7% ; 19.1 - 96.6%).This data shows the impact the selection of a type of fluorimeter may have on the comparability of test results of clinical samples. Selection of a measurement system with the lowest inter-laboratory variation in clinical studies shows a benefit for the comparability of study results.

Sensitive and selective spectrofluorimetric determination of chromium(VI) in water by fluorescence enhancement

A new fluorogenic method for the selective and sensitive determination of chromium(VI) in acidic water using rhodamine B hydrazide was developed. This method was based on the oxidation of non-fluorescent rhodamine B hydrazide by potassium dichromate in acidic aqueous conditions to give rhodamine B, which was highly fluorescent, as a product. With the optimum condition described, the fluorescence enhancement at 585 nm was linearly related to the concentration of chromium(VI) in the range of 5.0 × 10 −8 to 2.0 × 10 −6 mol L −1 (2.60–104 ng mL −1) with a correlation coefficient of R 2 = 0.9993 ( n = 18) and a detection limit of 5.5 × 10 −9 mol L −1 (0.29 ng mL −1). The R.S.D. was 2.2% ( n = 5). The proposed method was also applied to the determination of chromium(VI) in drinking water, river water and synthetic samples.

XIAP mRNA levels but not XAF1 or XIAP/XAF1 mRNA levels predict pathological response in bladder cancer patients treated with neoadjuvant chemotherapy

20030 Background: The relation between apoptosis-related molecules and chemosensitivity has been extensively studied. In recent years, attention has shifted to a new family of inhibitor of apoptosis proteins (IAPs). XIAP (X- linked inhibitor of apoptosis) is the most versatile and potent member of the IAP family. To date, the overexpression of XIAP has been detected in various cancers. XAF1 (X-linked inhibitor of apoptosis associated factor 1) is a new protein identified for its ability to interact with XIAP. Neither XIAP nor XAF1 or XIAP/XAF1 mRNA expression have been studied in bladder cancer patients. Methods: The expression of XIAP and XAF1 mRNA was analyzed by a real time quantitative fluorogenic PCR method in a group of 17 patients with locally advanced bladder cancer treated with a combination of neoadjuvant Gemcitabine and Cisplatin. The prognostic significance of XIAP and XAF1 mRNA expression and the correlation with several clinicopathological variables was evaluated. Results: XIAP and XAF1 mRNA expression was detected in all 17 (100%) case samples. The levels of XIAP mRNA expression showed a moderate variation among samples. In contrast, XAF1 and XIAP/XAF1 mRNA levels showed significant variation among samples. Bivariate correlation analyses revealed a significant positive Spearman direct correlation coefficient between the XIAP expression and the pathological response. No significant correlation was found for XAF1 expression as well as for the XIAP/XAF1 ratio and clinical and pathological response. Conclusions: This is first study to address the role of XIAP, its negative regulator XAF1, and the XIAP/XAF1 ratio in bladder cancer patients. The positive correlation between the XIAP mRNA expression and the pathological response is in line with a previous study from our group in which a correlation was found between XIAP expression and survival. All these observations point to a complex role of XIAP in tumor biology. XAF1 mRNA expression in bladder carcinomas did not achieve significance as an independent predictive and prognostic factor in a bivariate analysis. Further studies are necessary in order to better assess a possible clinical value for XIAP and XAF1 as predictive and prognostic markers in cancer patients. No significant financial relationships to disclose.

Characterization and Inhibition of SARS-Coronavirus Main Protease

Severe acute respiratory syndrome (SARS) is an emerging infectious disease caused by a novel human coronavirus (CoV). During the 2003 epidemic, the disease rapidly spread from its origin in southern China to other countries and affected almost 8000 patients, which resulted in about 800 fatalities. A chymotrypsin-like cysteine protease named 3C-like protease (3CLpro) is essential for the life cycle of the SARS-CoV. This main protease is responsible for maturation of functional proteins and represents a key anti-viral target. HPLC and fluorescence-based assays have been used to characterize the protease and to determine the potency of the inhibitors. The fluorogenic method monitoring the increase of fluorescence from the cleavage of a peptide substrate containing an Edans-Dabcyl fluorescence quenching pair at two ends has enabled the use of high throughput screening to speed up the drug discovery process. Several groups of inhibitors have been identified through high throughput screening and rational drug design approaches. Thus, alpha,beta-unsaturated peptidomimetics, anilides, metal-conjugated compounds, boronic acids, quinolinecarboxylate derivatives, thiophenecarboxylates, phthalhydrazide-substituted ketoglutamine analogues, isatin and natural products have been identified as potent inhibitors of the SARS-CoV main protease. The different classes of inhibitors reported in these studies are summarized in this review. Some of these inhibitors could be developed into potential drug candidates, which may provide a solution to combat possible reoccurrence of the SARS and other life-threatening viruses with 3CL proteases.

Exonuclease-released Fluorescence Detection of Human Parvovirus B19 DNA.

Background: The 5;-->3;-exonuclease activity of Thermus aquaticus (Taq) DNA polymerase permits polymerase chain reaction (PCR) product detection immediately after amplification using a fluorogenic probe. This approach eliminates the requirement for gel electrophoresis or enzyme immunoassays (EIA). The ligonucleotide probe is labeled with a reporter dye at its 5' terminus and a quencher dye at its 3' terminus and is present during DNA amplification. The exonuclease cleaves the reporter molecule from the probe-template hybrid, releasing it from the influence of the quencher molecule. The result is an increase in reporter fluorescence that can be read directly in a fluorescence spectormeter. In contrast to time-consuming gel electrophoresis and Southern blot hybridization or an EIA, this method can produce results from an entire 96-well microtiter plate in 15 minutes. Methods and Results: A B19-specific fluorogenic probe was synthesized containing a 5'-FAM label and a 3'-TAMRA label. Thirty clinical samples were analyzed for Human parvovirus B19 DNA by PCR amplification using both the fluorogenic and EIA method. Conclusions: Results generated with the fluorogenic probe correlated perfectly with those of the EIA, and the method would be particularly useful for high-volume work loads where gel or EIA-based approaches would be cumbersome.

Decomposition of macrophytes and dynamics of enzyme activities in subalpine marshes in Lake Tahoe basin, U.S.A.

To reveal the environmental and substrate quality effects on decomposition process and enzyme activities, litterbag experiments containing Nuphar and Carex leaves, Nuphar rhizome, and Ranunculus shoot, were carried in five-subalpine marshes in Lake Tahoe basin, USA. Alkaline phosphatase, β-glucosidase, and β-xylosidase activities were determined by a fluorogenic method using methyumbelliferyl substrates. Carex leaves, Nuphar rhizome and leaves, and Ranunculus shoots lost, respectively, 33, 67, 82 and 93% of original dry weight over 268 days. Decay rates were different among substrates but not among marshes. Nitrogen and carbon contents increased during the first 58 days and subsequently remained stable. Phosphorus content was stable during the experimental period except for a decrease in the first 16 days in Nuphar shoots. Enzyme activities in decomposing Carex and Nuphar leaves in four marshes were not significantly affected by environmental conditions. β-glucosidase and β-xylosidase activities in decomposing Carex leaves increased with time, but in other plant tissue these enzyme activities remained stable during experimental period. Enzyme activities were significantly different among decomposing substrates. Alkaline phosphatase activity was highest in Nuphar leaves (ca. 1286 µ-mole h−1 g DW −1) but lower and similar in other plant tissues (ca. 100 and 10 µ-mole h −1 g DW −1, respectively). This study showed differences in decay rates and enzyme activities rely on substrate and not the environment conditions of the study area. Decomposition rates in the early stage of decomposition were related to cumulative enzyme activities.

Establishment of fluorogenic quantitative method for the measurement of the B-lymphocyte stimulator gene expression

目的 研究建立人B细胞激活因子(BlyS)mRNA荧光定量检测方法,探讨正常人外周血单个核细胞(PBMC)中BlyS基因表达水平.方法基于TaqMan荧光探针技术,构建克隆载体pMD18-T-BlyS作为定量模板,在GeneAmp 5700型检测仪上,通过荧光强度达到一定阈值时的循环数来定量起始模板,以建立实时荧光定量逆转录聚合酶链反应(FQ-RT-PCR)方法;并对20名健康献血者外周血中BlyS表达进行检测.结果FQ-RT-PCR的动态检测范围为104～109拷贝/μg RNA(r≥0.996),内参三磷酸甘油醛脱氢酶的动态检测范围为103～108拷贝/μg RNA(r≥0.998);低值的批内、批间的重复性分别为17.7%和24.3%,高值的批内、批间的重复性分别为5.3%和8.1%;20名献血员健康外周血的PBMC中均有BlyS mRNA的表达,范围为5.5×104～4.9×105拷贝/μg,均值为(1.7±1.4)×105拷贝/μg.结论成功建立人BlyS基因表达FQ-PCR检测方法,检测结果定量准确可靠,可用于BlyS基因表达与自身免疫性疾病发病机制的关系研究。