采用竞争性定量PCR检测幽门螺杆菌cagA基因

目的建立竞争性定量 PCR 检测幽门螺杆菌 cagA 基因的方法。方法以重组 PCR 将乳糖操纵子中 Lac 阻抑蛋白特异性结合序列(21bp)重组入 cagA 基因397bp 的片段中构建内参标模援(rfcagA)。体外克隆并表达具有双功能的 GST-LacI 融合蛋白,其一端只有 GST 酶活性,另一端可特异性地与重组内参标模板结合。在定量 PCR 中,以 rfcagA 为内参标模板,pMC3 或 Hp 基因组 cagA 作为竞争性模板,PCR 产物的5′-端标记了生物素,可与包被了亲和素的微孔板结合,只有 rfcagA 的 PCR 产物可与融合蛋白结合而显色。结果 GST 底物的显色程度与样本的 ca-gA 含量呈负相关,当反应体系中的起始模板量为10-10~5拷贝,循环次数小于等于20次。原始模板数以指数方式增长,并建立了原始模板的考贝数与 A_(340)值同的标准曲线。用该方法检测12份已知菌株中的 cagA 含量,8份 cagA 阳性,cagA 基因的拷贝数为6.3×10~(10)-2.14×10~(11)/L,特异性为100%,敏感度达可检出 cagA 基因2倍的差别。结论这是一个特异、敏感及很实用的定量检测 cagA 基因的方法。     

Development and validation of InnoQuant™, a sensitive human DNA quantitation and degradation assessment method for forensic samples using high copy number mobile elements Alu and SVA

There is a constant need in forensic casework laboratories for an improved way to increase the first-pass success rate of forensic samples. The recent advances in mini STR analysis, SNP, and Alu marker systems have now made it possible to analyze highly compromised samples, yet few tools are available that can simultaneously provide an assessment of quantity, inhibition, and degradation in a sample prior to genotyping. Currently there are several different approaches used for fluorescence-based quantification assays which provide a measure of quantity and inhibition. However, a system which can also assess the extent of degradation in a forensic sample will be a useful tool for DNA analysts. Possessing this information prior to genotyping will allow an analyst to more informatively make downstream decisions for the successful typing of a forensic sample without unnecessarily consuming DNA extract. Real-time PCR provides a reliable method for determining the amount and quality of amplifiable DNA in a biological sample.Alu are Short Interspersed Elements (SINE), approximately 300 bp insertions which are distributed throughout the human genome in large copy number. The use of an internal primer to amplify a segment of an Alu element allows for human specificity as well as high sensitivity when compared to a single copy target. The advantage of an Alu system is the presence of a large number (>1000) of fixed insertions in every human genome, which minimizes the individual specific variation possible when using a multi-copy target quantification system. This study utilizes two independent retrotransposon genomic targets to obtain quantification of an 80 bp “short” DNA fragment and a 207 bp “long” DNA fragment in a degraded DNA sample in the multiplex system InnoQuant™. The ratio of the two quantitation values provides a “Degradation Index”, or a qualitative measure of a sample's extent of degradation. The Degradation Index was found to be predictive of the observed loss of STR markers and alleles as degradation increases. Use of a synthetic target as an internal positive control (IPC) provides an additional assessment for the presence of PCR inhibitors in the test sample.In conclusion, a DNA based qualitative/quantitative/inhibition assessment system that accurately predicts the status of a biological sample, will be a valuable tool for deciding which DNA test kit to utilize and how much target DNA to use, when processing compromised forensic samples for DNA testing.     

Toward Quantitative Small Animal Pinhole SPECT: Assessment of Quantitation Accuracy Prior to Image Compensations

Purpose  We assessed the quantitation accuracy of small animal pinhole single photon emission computed tomography (SPECT) under the current preclinical settings, where image compensations are not routinely applied. Procedures  The effects of several common image-degrading factors and imaging parameters on quantitation accuracy were evaluated using Monte-Carlo simulation methods. Typical preclinical imaging configurations were modeled, and quantitative analyses were performed based on image reconstructions without compensating for attenuation, scatter, and limited system resolution. Results  Using mouse-sized phantom studies as examples, attenuation effects alone degraded quantitation accuracy by up to −18% (Tc-99m or In-111) or −41% (I-125). The inclusion of scatter effects changed the above numbers to −12% (Tc-99m or In-111) and −21% (I-125), respectively, indicating the significance of scatter in quantitative I-125 imaging. Region-of-interest (ROI) definitions have greater impacts on regional quantitation accuracy for small sphere sources as compared to attenuation and scatter effects. For the same ROI, SPECT acquisitions using pinhole apertures of different sizes could significantly affect the outcome, whereas the use of different radii-of-rotation yielded negligible differences in quantitation accuracy for the imaging configurations simulated. Conclusions  We have systematically quantified the influence of several factors affecting the quantitation accuracy of small animal pinhole SPECT. In order to consistently achieve accurate quantitation within 5% of the truth, comprehensive image compensation methods are needed. Significance: We assessed the quantitation accuracy of small animal pinhole SPECT, providing reference information for the current preclinical studies.     

Clinical diagnosis of early dengue infection by novel one-step multiplex real-time RT-PCR targeting NS1 gene

BackgroundDengue is a mosquito-borne disease that causes a public health problem in tropical and subtropical countries. Current immunological diagnostics based on IgM and/or nonstructural protein 1 (NS1) antigen are limited for acute dengue infection due to low sensitivity and accuracy.ObjectivesThis study aimed to develop a one-step multiplex real-time RT-PCR assay showing higher sensitivity and accuracy than previous approaches.Study designSerotype-specific primers and probes were designed through the multiple alignment of NS1 gene. The linearity and limit of detection (LOD) of the assay were determined. The assay was clinically validated with an evaluation panel that was immunologically tested by WHO and Malaysian specimens.ResultsThe LOD of the assay was 3.0 log₁₀ RNA copies for DENV-1, 2.0 for DENV-3, and 1.0 for DENV-2 and DENV-4. The assay showed 95.2% sensitivity (20/21) in an evaluation panel, whereas NS1 antigen- and anti-dengue IgM-based immunological assays exhibited 0% and 23.8–47.6% sensitivities, respectively. The assay showed 100% sensitivity both in NS1 antigen- and anti-dengue IgM-positive Malaysian specimens (26/26). The assay provided the information of viral loads and serotype with discrimination of heterotypic mixed infection.ConclusionsThe assay could be clinically applied to early dengue diagnosis, especially during the first 5 days of illness and approximately 14 days after infection showing an anti-dengue IgM-positive response.     

Quantifying trifluoroacetic acid as a counterion in drug discovery by 19F NMR and capillary electrophoresis

Drug discovery compounds are often isolated as salts of trifluoroacetate from preparative high performance liquid chromatography, which are then used for biological assays in order to assess their efficacy against the biochemical target of interest. It is, therefore, imperative to determine the TFA content in order to ascertain the correct formula weight and when required, to ensure that the TFA has been completely exchanged for another counterion in order to have superior pharmacokinetic properties and to avoid potential toxicity effects. In this paper, we present capillary electrophoresis and (19)F nuclear magnetic resonance methods for determining the TFA content of drug discovery compounds. Furthermore, these methods have been successfully applied in a high-throughput fashion, which is a key feature for general applicability in a pharmaceutical setting.     

弥散张量成像量化分析脑胶质瘤侵袭性*☆

背景：胶质瘤侵袭能力的量化对于指导治疗或筛检肿瘤侵袭因子均有重要意义。在实验研究中有较为成熟的方法进行量化,但尚无很好的临床量化胶质瘤侵袭力的方法。 目的：应用磁共振弥散张量成像量化分析脑胶质瘤的侵袭性。 设计、时间及地点：量化分析,于2005-10/2007-01在重庆医科大学附属第一医院神经外科、放射科、病理科,解放军第三军医大学附属西南医院放射科完成。 对象：选择幕上脑胶质瘤患者20例,男14例,女6例;年龄19~73岁,平均51.3岁。 方法：全部患者均进行手术治疗,术中行内减压,获取瘤周水肿区的组织标本,病理检查作肿瘤细胞计数和细胞密度分析,建立肿瘤脑浸润程度的病理标准。 主要观察指标：利用磁共振弥散张量成像测量肿瘤实体区、瘤周水肿区、水肿外区、相对正常白质区和对侧白质区5个兴趣区的分量各向异性值和表观平均弥散系数值;将不同兴趣区的分量各向异性值和表观平均弥散系数值与肿瘤病理脑浸润程度作相关分析。 结果：①肿瘤分量各向异性值由瘤体区向外呈递增趋势,依次为0.055±0.017,0.107±0.037,0.255±0.064,0.376±0.070,0.396±0.099,其中最大增幅出现在瘤周水肿区和水肿外区之间。②瘤周水肿区的分量各向异性值与胶质瘤病理脑浸润程度呈负相关性,表观平均弥散系数值在瘤周水肿区明显增高,但在其他区域的增减变化无规律。 结论：瘤周水肿区分量各向异性值可作为脑胶质瘤侵袭性的量化指标。     

抗-HCV阳性血清HCV RNA检测与基因分型的研究

目的 检测抗-HCV阳件血清巾的HCV RNA并进行HCV基因分型.方法 采用荧光定量PCR法检测85例大连地区抗-HCV阳性患者血清中HCV RNA,应用型特异性引物逆转录套式PCR法对HCV RNA阳性样本进行基因分型.结果 85例的抗-HCV阳性患者中,HCV RNA阳性65例(76.5%),其中基因分型1b型32例(49.2%),2a型29例(44.6%),未分型4例(6.2%).结论 抗-HCV阳性并非HCV直接标志,大连地区HCV基冈1b型和2a型基本相等.     

Development and validation of a liquid chromatography–tandem mass spectrometry assay for the simultaneous quantitation of prednisolone and dipyridamole in human plasma and its application in a pharmacokinetic study

We have developed and validated an accurate, sensitive, and robust LC–MS/MS method that determines the concentration of CRx-102 (the combination of prednisolone and dipyridamole) in human plasma. In this method, prednisolone, dipyridamole, and the combined internal standards (IS) prednisolone-d₆ (IS for prednisolone) and dipyridamole-d₂₀ (IS for dipyridamole) were extracted from 100 μL human EDTA plasma using methylbutyl ether. Calibration curves were linear over a concentration range of 0.4–200 ng/mL for prednisolone and 5–3000 ng/mL for dipyridamole. The analytes were quantitatively determined using tandem mass spectrometry operated in positive electrospray ionization in a multiple reaction monitoring (MRM) mode. This validated method has been used successfully in clinical pharmacokinetic studies of CRx-102 in healthy volunteers.     

Variations in Mitochondrial DNA Copy Numbers in MS Brains

The aim of this study is to determine if there is a pathology-related variation in mitochondrial (mt)DNA copy numbers in brains of patients with multiple sclerosis (MS). Our recent study demonstrated an age-dependent but excluded a MS pathology-related increase in the proportion of cytochrome oxidase (COX)-negative cells and deleted mtDNA molecules in postmortem brain tissue specimens of patients and controls (Blokhin et al., Neuromolecular Medicine, in press, 2008). This corollary study further extends our efforts defining mitochondrial contributions to tissue degeneration associated with inflammatory demyelination. Copy number variations of mtDNA molecules were defined by quantifying the mtDNA ND1 gene copies relative to the invariable nuclear ribosomal 18S gene copies (ND1/r18S) using real-time polymerase chain reaction analyses in laser dissected, COX-positive and COX-negative single neurons and glial cells from frozen postmortem normal-appearing gray (NAGM) and white matter (NAWM) regions and chronic active plaques of MS patients, and gray matter (GM) and white matter (WM) regions of age matched non-neurological disease (NND) controls. ND1/r18S values were correlated with tissue regions, pathology, and age. While the ND1/r18S values were similar in NAWM and plaque-containing specimens of MS patients as well as in NAWM of patients and WM of age-matched NND controls, we found significantly higher mtDNA copy number values in neurons of NAGM than in cells of other MS brain regions. The ND1/r18S values were even higher in NAGM than in GM of age-matched NND controls. An age-related decline in ND1/r18S values was also noted in neurons of both MS patients and NND controls. These observations exclude a change in mtDNA copy numbers in plaques, however, suggest a compensatory replication of mtDNA or mitochondria in the cortex with neuroaxonal loss in MS. The age-related decline in mtDNA copy numbers may explain some features of late-onset MS.