Prenatal diagnosis by enzymatic amplification and restriction endonuclease digestion for detection of haemoglobins A, S and C.

The detection of the single base pair mutations at codon 6 of the beta-globin gene is important for the prenatal diagnosis of sickle-cell anaemia and SC disease. A novel procedure has been designed to create a restriction site at both the beta A and beta C alleles to facilitate the discrimination of haemoglobins A, S and C. The general principle of this procedure is to enzymatically amplify genomic DNA using a modified primer containing an altered 3'-terminal nucleotide to create these restriction sites. After this modified primer has been efficiently incorporated into amplified DNA, the PCR products are digested with the restriction enzymes Ava I and Sty I. Ava I recognizes a site in amplified DNA containing a beta A allele, and Sty I recognizes a site in DNA containing a beta C allele. Since the beta A and beta C alleles can be distinguished directly by the presence of a restriction site, the beta S allele can be identified indirectly. All three beta-globin alleles are easily distinguished by size and pattern of electrophoresed fragments on agarose gels. This procedure is specific and sensitive, thus permitting rapid, economical diagnosis of sickle-cell anaemia and SC disease.     

NMR-based metabolomics with enhanced sensitivity

NMR-based metabolomics, which emerged along with mass spectrometry techniques, is the preferred method for studying metabolites in medical research and food industries. However, NMR techniques suffer from inherently low sensitivity, regardless of their superior reproducibility. To overcome this, we made two beneficial modifications: we detuned the probe to reach a position called “Spin Noise Tuning Optimum” (SNTO), and we replaced the conventional cylindrical 5 mm NMR tube with an electric field component-optimized shaped tube. We found that concerted use of both modifications can increase the sensitivity (signal to noise ratio per unit volume) and detection of metabolites and decrease the measurement time by order of magnitude. In this study, we demonstrate and discuss the achieved signal enhancement of metabolites on model non-human (bovine serum, amino acid standard mixture) and human urine samples.

We combined Spin Noise Tuning Optimum (SNTO) and electric field component-optimized shaped tube to boost sensitivity for NMR-based metabolomics.     

纳米金标记银染增强法快速检测痰液结核分枝杆菌

目的建立快速检测结核分枝杆菌(MTB)的纳米金标记银染增强法(金标记银染法)。方法以MTB的IS6110基因为靶基因,分别与互补的纳米金探针、生物素探针杂交,形成三明治杂交结构,然后借链霉亲合素将靶序列锚定在酶标孔内,通过纳米金催化的银染放大效应产生高灵敏的识别信号,在酶联仪上检测其吸光度(A)值。用本法、传统固体培养法、PCR法检测90例可疑患者痰标本并进行比对。结果金标记银染法检测限达1 pmol/L,90例临床标本的MTB检出率与PCR法均为42.2%(38/90);培养法MTB检出率为36.7%(33/90),金标记银染法与其检测结果差异无统计学意义(P0.05)。结论成功构建了金标记银染法,可直接应用于临床疑似肺结核患者痰标本的快速检测,具有简便快速、灵敏度高、特异性强等优点。     

Rapid and sensitive detection of Enterobacter sakazakii by cross-priming amplification combined with immuno-blotting analysis

Enterobacter sakazakii is a widespread and life-threatening bacterium especially in polluted powdered infant milk formula. Several methods have been developed for detection of E. sakazakii such as physiological and biochemical methods, PCR and loop-mediated isothermal amplification. However, these procedures were disadvantages due to a long assay time, low sensitivity or the use of toxic reagents. Our method of cross-priming amplification (CPA) under isothermal conditions combined with immuno-blotting analysis made the whole detection procedure more sensitivity and lower time-consuming. A set of specific displacement primers, cross primers and testing primers were designed based on six specific sequences in E. sakazakii 16S-23S rDNA internal transcribed spacer. Under isothermal condition at 63 °C for 60 min, the specific amplification and hybridization steps were processed simultaneously. The specificity of the CPA was tested in panel of 54 different bacterial strains and 236 milk powder products. Two red signal lines were developed on the BioHelix Express strip in all of positive E. sakazakii strains, and only one signal line was demonstrated by non- E. sakazakii bacterial strains. The limit of decetion of CPA was 6.3 ± 2.7277 fg for the genomic DNA, 88 ± 8.7892 cfu/ml for pure bacterial culture, and 3.2 ± 2.0569 cfu per 100 g milk powder with pre-enrichment. The current study demonstrated that the assay method of CPA combined with immuno-blotting analysis was a specific and sensitive detection for the rapid detection of E. sakazakii.     

Quantification of the detection of Pneumocystis carinii by DNA amplification.

We have developed a highly specific and sensitive technique for the detection of Pneumocystis carinii DNA using DNA amplification by the polymerase chain reaction (PCR). PCR products are detected by agarose gel electrophoresis and Southern hybridization to an oligonucleotide probe. Here we report the calibration of parasite numbers with amplification and hybridization signals and show that we can detect P. carinii to a lower limit of one to two organisms. The quantification of this diagnostic technique allows us to establish the number of organisms in a clinical sample which correspond to pneumocystis pneumonia or to sub-clinical pulmonary colonization.     

Simplified sodium dodecyl sulfate-polyacrylamide gel electrophoresis of unconcentrated urine with enhanced resolution and detection sensitivity

We applied a simple sodium dodecyl sulfate-polyacrylamide gel electrophoresis method to urine. The method, developed for serum protein analysis (Clin Chem 1984;30:475-9), has a high sample throughput and gives excellent resolution with unconcentrated urine. It clearly distinguishes and characterizes proteinuric urine (7.5 microL) by Coomassie Blue staining and gives complex silver-stained patterns with nonproteinuric urine (2 microL). The former is recommended for routine clinical screening, the latter for research purposes.     

Capillary and microchip electrophoresis for rapid detection of known mutations by combining allele-specific DNA amplification with heteroduplex analysis

BACKGROUND: Detection of mutations by gel electrophoresis and allele-specific amplification by PCR (AS-PCR) is not easily scaled to accommodate a large number of samples. Alternative electrophoretic formats, such as capillary electrophoresis (CE) and microchip electrophoresis, may provide powerful platforms for simple, fast, automated, and high-throughput mutation detection after allele-specific amplification. METHODS: DNA samples heterozygous for four mutations (185delAG, 5382insC, 3867G-->T, and 6174delT) in BRCA1 and BRCA2, and homozygous for one mutation (5382insC) in BRCA1 and two mutations (16delAA and 822delG) in PTEN were chosen as the model system to evaluate the capillary and microchip electrophoresis methods. To detect each mutation, three primers, of which one was labeled with the fluorescent dye 6-carboxyfluorescein and one was the allele-specific primer (mutation-specific primer), were used to amplify the DNA fragments in the range of 130-320 bp. AS-PCR was combined with heteroduplex (HD) analysis, where the DNA fragments obtained by AS-PCR were analyzed with the conditions developed for CE-based HD analysis (using a fluorocarbon-coated capillary and hydroxyethylcellulose). The CE conditions were transferred into the microchip electrophoresis format. RESULTS: Three genotypes, homozygous wild type, homozygous mutant, and heterozygous mutant, could be identified by CE-based AS-PCR-HD analysis after 10-25 min of analysis time. Using the conditions optimized with CE, we translated the AS-PCR-HD analysis mutation detection method to the microchip electrophoresis format. The detection of three heterozygous mutations (insertion, deletion, and substitution) in BRCA1 could be accomplished in 180 s or less. CONCLUSIONS: It is possible to develop a CE-based method that exploits both AS-PCR and HD analysis for detecting specific mutations. Fast separation and the capacity for automated operation create the potential for developing a powerful electrophoresis-based mutation detection system. Fabrication of multichannel microchip platforms may enable mutation detection with high throughput.     

Operant conditioning of enhanced pain sensitivity by heat-pain titration

Operant conditioning mechanisms have been demonstrated to be important in the development of chronic pain. Most experimental studies have investigated the operant modulation of verbal pain reports with extrinsic reinforcement, such as verbal reinforcement. Whether this reflects actual changes in the subjective experience of the nociceptive stimulus remained unclear. This study replicates and extends our previous demonstration that enhanced pain sensitivity to prolonged heat-pain stimulation could be learned in healthy participants through intrinsic reinforcement (contingent changes in nociceptive input) independent of verbal pain reports. In addition, we examine whether different magnitudes of reinforcement differentially enhance pain sensitivity using an operant heat-pain titration paradigm. It is based on the previously developed non-verbal behavioral discrimination task for the assessment of sensitization, which uses discriminative down- or up-regulation of stimulus temperatures in response to changes in subjective intensity. In operant heat-pain titration, this discriminative behavior and not verbal pain report was contingently reinforced or punished by acute decreases or increases in heat-pain intensity. The magnitude of reinforcement was varied between three groups: low (N1=13), medium (N2=11) and high reinforcement (N3=12). Continuous reinforcement was applied to acquire and train the operant behavior, followed by partial reinforcement to analyze the underlying learning mechanisms. Results demonstrated that sensitization to prolonged heat-pain stimulation was enhanced by operant learning within 1h. The extent of sensitization was directly dependent on the received magnitude of reinforcement. Thus, operant learning mechanisms based on intrinsic reinforcement may provide an explanation for the gradual development of sustained hypersensitivity during pain that is becoming chronic.     

Assessment of pan-Leishmania detection by recombinase polymerase amplification assay

The spread of vector habitats along with increasing human mobility can introduce atypical Leishmania species and hence can challenge existing diagnostic practices for rapid detection of active infection with species outside the narrow target range. Here we assessed the pan-Leishmania detection ability of isothermal recombinase polymerase amplification (RPA) assays targeting 18S rRNA gene, cathepsin L-like cysteine proteinase B (Cpb) gene, and kinetoplast minicircle DNA (kDNA) regions. While the lowest limit of detection of the 18S rRNA-RPA and Cpb-RPA assays were estimated as 12 and 17 standard DNA molecules, respectively, both assays could amplify genomic DNA of 7 pathogenic Leishmania species. Evaluation of 18S rRNA-RPA and our previously developed kDNA-RPA assays on 70 real-time PCR-positive leishmaniasis samples of varying pathologies resulted in sensitivity rates of 35.71% and 88.57%, respectively, while the combined sensitivity was 98.57%. Combinatorial application of 18S rRNA-RPA and kDNA-RPA assays can be recommended for further diagnostic assessments.     

Rapid on-site detection of Acidovorax citrulli by cross-priming amplification

Cross-priming amplification (CPA) for Acidovorax citrulli detection was evaluated in this study. The sensitivity of CPA assay for pure bacterial culture was 3.7 × 10(3) CFU/ml. Bacteria on naturally infected watermelon seeds were detected using CPA assay, suggesting this method is suitable for A. citrulli on-site detection from watermelon seeds.     

Immunophenotypic analysis in the diagnosis of malignant lymphoma

Immunophenotypic analysis is capable of distinguishing benign and malignant or determining the lineage of neoplastic cells in most of lymphoproliferative disorders. The methodological approaches can be divided into two major categories; immunohistochemistry and flow cytometry. Both of these techniques can be important adjuncts to histological diagnosis although flow cytometry is not yet common in Japan. The basic features to be checked by immunophenotyping are as follows. IPT-I: Light chain restriction of immunoglobulin. IPT-II: Loss or stronger expression of one or more antigens normally expressed in reactive lymphocytes. IPT-III: Significant increase of a particular cell population that is quite rare or absent from normal tissue at the biopsy site. While IPT-I could be taken as direct evidence of a clonal proliferation of B-lineage cell, IPT-II and -III are the only strong suggestions of a malignant lymphoid proliferation.     

Evaluation of chimerism in DNA samples by PCR amplification of D1S80 with detection by capillary electrophoresis.

BACKGROUND: Analysis of the relative amounts of donor and recipient DNA in bone marrow after bone marrow transplantation is frequently used to determine the status of the transplant. We studied the performance of an assay to quantify chimerism based on amplification of the D1S80 variable number tandem repeat marker by PCR with detection of PCR products by capillary electrophoresis (CE). METHODS AND RESULTS: Samples from potential bone marrow donors and recipients were analyzed separately and in mixtures to simulate various degrees of chimerism from 10% to 90% and subjected to PCR/CE analysis. There was excellent agreement between the measured and known relative proportions of DNA components in chimeric samples. The lower limit of sensitivity for detection of chimerism was 1%; between-runs coefficients of variation were <5%. CONCLUSIONS: Amplification of the D1S80 minisatellite by PCR with CE detection is a reliable method for determination of the relative contribution of different DNAs in mixed samples. This method is fast, quantitative, and extremely reproducible.     

71例急性早幼粒细胞白血病患者白血病细胞免疫表型分析

本研究通过回顾性分析急性早幼粒细胞白血病(APL)患者骨髓异常早幼粒细胞的免疫表型及患者初诊资料,探讨其免疫表型特点及其意义。利用常规6色免疫分型方法对71例APL患者的白血病细胞进行免疫表型分析。结果发现:MPO、CD33和CD13在所有患者的APL细胞中都有较强表达,其平均阳性细胞比例达到88%以上。CD117的阳性表达率为50.7%,其平均阳性细胞比例为52.5%。约10%患者的白血病细胞表达CD15,但大部分病例的阳性细胞率都集中在20%-40%的弱表达范围内,其平均阳性细胞比例为42.5%。少数患者的异常细胞表达CD34和HLA-DR,且表达强度较弱。约25%患者的APL细胞跨系表达了CD2、CD56,大部分也都集中于20%-60%的低表达范围内,其平均阳性细胞比例分别为39.3%和42.3%。由此认为,APL的典型免疫表型为MPO+CD13+CD33+CD117±CD15±CD34-HLA-DR-。CD2和CD56在CD34+或HLA-DR+组(包括CD34+HLA-DR+、CD34+HLA-DR-和CD34-HLA-DR+)的阳性比例明显高于CD34-和HLA-DR-组。初诊患者外周血白细胞计数、血小板计数、外周血中异常早幼粒细胞比例及CD13的阳性比例在CD15<10%、10%20%3组均出现显著的统计学差异。结论:APL患者的异常早幼粒细胞的免疫表型具有独特的特征,多色流式细胞术检测可辅助APL的快速诊断,对分析白血病细胞的来源和判断患者预后亦可能有着重要意义。     

One-pot detection of telomerase activity with high sensitivity and specificity via RNA FRET probes and RNase H-assisted signal cycling amplification

Human telomerase is a universal cancer biomarker and a promising anticancer therapeutic target. Sensitive and specific detection of telomerase activity is of great significance for cancer diagnosis and treatment. Up to now, many methods have been established to detect the activity of telomerase, but most of these methods require complex probe design and tedious experimental steps generally including telomere extension reaction, amplification of the extended products and signal detection. Herein, we propose a one-pot method to detect the telomerase activity via RNA FRET probes and RNase H-assisted signal cycling amplification, and the proposed assay can integrate the telomere extension reaction, signal amplification and readout in one step without requirement of amplification of the extended products, which greatly simplifies the experimental design and operation steps. Additionally, the proposed one-pot method has high sensitivity and can unequivocally detect the telomerase activity in as few as 5 cancer cells, which holds great potential in telomerase-related fundamental and clinical studies.

A one-pot method is developed for the detection of telomerase activity via RNA FRET probes and RNase H-assisted signal cycling amplification.     

环介导等温扩增技术快速检测结核分枝杆菌核酸

摘要:目的：建立一种快速准确的检测结核分枝杆菌（MTB）核酸的环介导等温扩增（LAMP）方法。 方法：以重复插入序列IS6110为目的基因,设计LAMP引物,特异检测MTB核酸。用本法与痰涂片抗酸染色镜检法、实时荧光PCR法对100例可疑患者痰标本进行对比检查。 结果：LAMP法特异性强,仅扩增MTB复合群核酸;灵敏度高,检测限达100 fg;而实时荧光PCR检测限为1 pg。对100例疑似结核病患者痰液标本检测,涂片抗酸染色法、LAMP法、实时荧光PCR法的阳性率分别为28%、39%和38%。 结论：本研究建立的LAMP方法检测MTB核酸特异性强、灵敏度高、时间短且操作简便,有望成为临床快速检测MTB的新方法。     

环介导等温扩增技术快速检测副溶血性弧菌

目的建立一种检测副溶血性弧菌（Vp）快速且特异的环介导等温扩增（LAMP）检测方法。方法针对副溶血性弧菌不耐热溶血素（tlh）基因特异性序列的6个位点设计4条LAMP引物,65℃保温约60min,完成对副溶血性弧菌的扩增,扩增产物经肉眼、SYBR Green Ⅰ染色、电泳和酶切鉴定。利用LAMP和普通PCR方法同时检测1株副溶血性弧菌和12株非副溶血性弧菌来验证LAMP方法的特异性;将副溶血性弧菌菌液作一系列10倍稀释后用LAMP和PCR方法进行检测,比较两者敏感性。结果1株副溶血性弧菌出现LAMP扩增反应：通过肉眼、SYBR Green Ⅰ染色和电泳均能观察到LAMP扩增产物的出现,酶切证实了LAMP产物的特异性;12株非副溶血性弧菌未出现LAMP扩增。LAMP检测tlh基因的特异性和敏感性与普通PCR相同,LAMP检测tlh基因的检测下限为15cfu／mL。结论建立了一种快速、敏感、特异的副溶血性弧菌检测方法,适合日常监测及快速检测的需要。     

Flow-injection analysis with chemiluminescence detection.

In "flow-injection analysis," the sample is inserted into a stream of reagent by use of a sample-injection valve. Mixing occurs downstream from the valve in a coil of tubing. With chemiluminescence detection this coil is positioned in front of a photomultiplier. We have evaluated the system for detection of hydrogen peroxide, using the luminol reaction with cupric ion as a catalyst. The effects of flow rate, sample volume, and reaction kinetics on the magnitude, duration, and repeatability of the chemiluminescent response have been evaluated. Precisions of 1 to 2% relative standard deviation on replicate measurements are readily achievable. A sample throughput of six samples per minute is possible with very little peak overlap. This detection system can be coupled to any process in which peroxide is generated.