猪细胞因子SYBR Green实时PCR检测方法的建立

目的:建立一种用SYBR Green荧光染料检测猪细胞因子的实时PCR方法。方法:从猪外周血淋巴细胞中提取细胞因子mRNA,反转录成cDNA,利用SYBR Green荧光染料法实时检测IL8、IL10、IFNα、IFN(?)和TNFα的mRNA表达水平。分别通过融解曲线和琼脂糖凝胶电泳分析各细胞因子检测的特异性;并对各细胞因子PCR产物进行梯度稀释后作为模板进行敏感性试验;利用建立的方法分别对正常猪和感染PRRSV的猪的外周血淋巴细胞中上述细胞因子进行检测,并以管家基因cyclophilin为内参照对各细胞因子进行定量分析。结果:建立的各细胞因子SYBR Green实时检测方法具有良好的特异性和敏感性,TNFα检测敏感性可达10个拷贝,其它细胞因子检测敏感性均可达100个拷贝。猪在感染PRRSV后,外周血淋巴细胞中IL8和IL10明显增加,而IFNα和TNFα略有下降。结论:建立了五种猪细胞因子SYBR Green实时PCR检测方法,并能成功地应用于临床检测。     

Quantitative real-time RT-PCR using hybridization probes and imported standard curves for cytokine gene expression analysis

Quantitative real-time or kinetic RT-PCR is increasingly used for the quantification of specific mRNA targets, especially in clinical applications. To quantify the mRNA of cytokines and their receptors, which play important roles in the pathogenesis of autoimmune diseases such as multiple sclerosis, we have developed quantitative two-step RT-PCR assays for IL-4, IL-4R, IFN-gamma, IFN-beta, and the housekeeping gene porphobilinogen deaminase (PBGD). The LightCycler system was used to quantify the copy numbers with the sequence-specific hybridization probe detection format. The quantification was carried out on the basis of standard curves generated with external homologous plasmids for each different parameter in relation to the gene expression of PBGD. Therefore, this procedure represents a relative quantification method with external standards, as the standard curves were used to obtain an absolute value for the copy numbers of the targets and the reference (PBGD). The new software version 3.5 of the LightCycler system allows the construction of a single parameter-dependent plasmid standard curve for the quantification of unknown samples from different runs. Here we demonstrate how to achieve precise and reproducible quantification, even when using measurements from different PCR runs.     

A SYBR Green RT-PCR assay in single tube to detect human and bovine noroviruses and control for inhibition

Background

A subset of the virus-specific CD8+ cytotoxic T lymphocytes (CTL) isolated from the lungs of mice infected with human respiratory syncytial virus (RSV) is impaired in the ability to secrete interferon γ (IFNγ), a measure of functionality. It was suggested that the impairment specifically suppressed the host cellular immune response, a finding that could help explain the ability of RSV to re-infect throughout life.

Results

To determine whether this effect is dependent on the virus, the route of infection, or the type of infection (respiratory, disseminated, or localized dermal), we compared the CTL responses in mice following intranasal (IN) infection with RSV or influenza virus or IN or intradermal (ID) infection with vaccinia virus expressing an RSV CTL antigen. The impairment was observed in the lungs after IN infection with RSV, influenza or vaccinia virus, and after a localized ID infection with vaccinia virus. In contrast, we observed a much higher percentage of IFNγ secreting CD8+ lymphocytes in the spleens of infected mice in every case.

Conclusion

The decreased functionality of CD8+ CTL is specific to the lungs and is not dependent on the specific virus, viral antigen, or route of infection.     

大鼠尿激酶SYBR Green I real time RT-PCR引物的设计

设计用于SYBR Green I法实时定量逆转录多聚酶链反应(QRT-PCR)检测大鼠尿激酶型纤溶酶原激活因子(uPA)mRNA的引物。从基因库获取靶基因及相关序列,充分收集争分析相关生物信息学数据,应用Oligo 6.22设计出一对长度为21bp的引物,其GG含量为52.4%;上下游引物3’最稳定二聚体和及发夹结构的能量分别为-1.5、-0.40 kcal/mol和-3.5、-O.90 kcal/mol,引物间最稳定二聚体为-3.1 kcal/mol。5’端和中间△G值较高,高于3’端△G;引发效率分别455和403。实验证明,该引物能够高效、特异地实现对靶序列的检测,适用于SYBR Green I法实时定量检测(uPA)mRNA。     

SYBR Green I荧光RT-PCR检测犬瘟热方法的建立和应用

根据GenBank发表的犬瘟热病毒（CDV）的F基因序列,经过分析在F片段的保守区域内设计引物,建立了SYBR Green I荧光RT-PCR检测CDV的方法,并通过对厦门市宠物医院收集的临床发病和疑似发病的犬病料（包括眼分泌物、鼻拭子、唾液、血液、尿液等）的检测,结果表明,本研究建立的快速检测CDV的SYBR Green I荧光RT-PCR方法具有特异性强、灵敏度高、操作简便等优点,值得推广应用。     

Development of a SYBR Green I real-time PCR for quantitative detection of Vibrio alginolyticus in seawater and seafood

AIM: Vibrio alginolyticus is an economically important micro-organism. The main aim of the present study was to develop a real-time polymerase chain reaction (PCR) assay for rapid, sensitive and effective quantification of V. alginolyticus in seawater and seafood. METHODS AND RESULTS: Purified DNA of V. alginolyticus, artificially inoculated seawater and seafood tissue homogenates were subjected to the gyrB-targeted real-time PCR assay. Natural seawater and seafood samples were analysed by this real-time PCR protocol. Specificity tests showed that positive result was obtained only with V. alginolyticus strains. The detection sensitivity was determined to be 0.4 pg of genomic DNA equivalent to 72 cells per PCR in pure culture and 100 cells in 1 ml of seawater or seafood tissue homogenates. Single cell detection is achieved after 3 h of sample enrichment. CONCLUSIONS: A sensitive and specific SYBR Green I-based real-time PCR assay targeting gyrB gene was successfully developed to quantify V. alginolyticus within 6 h in seawater and seafood samples. SIGNIFICANCE AND IMPACT OF THE STUDY: No report on the molecular-based method was available for quantitative detection of V. alginolyticus. This work will provide a novel method for evaluation of the risk of V. alginolyticus to marine environmental health and seafood safety.     

SYBR GreenⅠ荧光RT-PCR检测犬瘟热方法的建立和应用

根据GenBank发表的犬瘟热病毒(CDV)的F基因序列,经过分析在F片段的保守区域内设计引物,建立了SYBR GreenⅠ荧光RT-PCR检测CDV的方法,并通过对厦门市宠物医院收集的临床发病和疑似发病的犬病料(包括眼分泌物、鼻拭子、唾液、血液、尿液等)的检测,结果表明,本研究建立的快速检测CDV的SYBR GreenⅠ荧光RT-PCR方法具有特异性强、灵敏度高、操作简便等优点,值得推广应用.     

Development and evaluation of a real-time quantitative PCR assay for Aspergillus flavus

Aspergillus flavus is a ubiquitous mold and the most common mold contaminating foodstuffs. Many strains of A. flavus produce aflatoxins. In addition it is an allergen and an opportunistic pathogen of animals and plants. A. flavus often is underestimated in traditional culture analyses due to the expertise required and the cost associated with speciating members of the genus Aspergillus. The goal of this study was to develop and validate a primer and probe set for the rapid detection and quantitation of A. flavus in pure culture using real-time quantitative polymerase chain reaction (QPCR) amplification. Unique DNA regions were located in the genome of the target organism by sequence comparison with the GenBank database, and several candidate oligonucleotides were identified from the scientific literature for potential use with the TaqMan QPCR technology. Three primer and probe sets were designed and validated for specificity and sensitivity in laboratory experiments. Initial screening to test for sensitivity was performed with seven A. flavus isolates and selected nontarget fungi. Specificity testing was conducted with the selected primer and probe set, which amplified all nine A. flavus isolates tested, including an aflatoxin producing strain. The primers did not amplify DNA extracted from 39 other fungal species (comprising 16 genera), including 18 other Aspergillus species and six Penicillium species. No amplification of human or bacterial DNA was observed; however cross-reactivity was observed with Aspergillus oryzae. PCR analysis of DNA dilutions and the use of an internal positive control demonstrated that 67% of the fungal DNA samples assayed contained PCR inhibitors. The assay validated for the target organism is capable of producing PCR results in less than 1 h after DNA extraction. The results of this research demonstrate the capabilities of QPCR for the enhanced detection and enumeration of fungi of significance to human health.     

A real-time PCR assay for the detection and quantitation of Campylobacter jejuni using SYBR Green I and the LightCycler

Campylobacter jejuni is recognized as a leading human food-borne pathogen. Traditional biochemical identification for C. jejuni is not reliable due to special growth requirements and the possibility that this bacterium can enter a viable but nonculturable (VNC) state. Nucleic acid-based tests have emerged as a useful alternative to traditional testing. In this article, we present fluorescent quantitative PCR assay for quantitative detection of C. jejuni, the assay was carried out using a LightCycler instrument and product formation was monitored continuously with the fluorescent double-stranded DNA binding dye SYBR Green I. When this assay was applied, the assay positive for all of the isolates of C. jejuni tested (11 isolates, including type strain ATCC33560) and negative for all other Campylobacter spp. (three isolates) and several other bacteria (five species tested). The total assay could be completed in 60 min with a detection limit of approximately 1 CFU, and a correlation coefficient was 1.000. Result indicated that fluorescent quantitative detection methods provided a special, sensitive, rapid, reproducible and accurate method for quantitative detection of C. jejuni.     

土壤中棉花黄萎病菌SYBR Green Ⅰ荧光RT-PCR定量检测技术研究

为实现田间土壤棉花黄萎病菌的早期检测,建立了土壤中棉花黄萎病菌的SYBR GreenⅠ荧光定量PCR检测方法.以含342bp PCR扩增产物的阳性质粒为参考,构建了标准曲线,并对该曲线的特异性、敏感性、可重复性进行了评价.结果表明,该方法具有快速、特异性强、敏感度高等特点.检测范围在3.8×103-3.8×108cop...     

Quantitation of rabbit cytokine mRNA by real-time RT-PCR

Fundamental understanding of rabbit immunology and the use of the rabbit as a disease model have long been hindered by the lack of immunological assays specific to this species. In the present study, we sought to develop a method to quantitate cytokine expression in rabbit cells and tissues. We report the development of a quantitative real-time RT-PCR method for measuring the relative levels of rabbit IFN-gamma, IL-2, IL-4, IL-10 and TNF-alpha mRNA. Quantitation was accomplished by comparison to a standard curve generated using plasmid DNA containing partial sequences of the relevant cytokines. Experimental studies demonstrate applicability of this assay to quantitate cytokine mRNA levels from rabbit spleen cells following mitogen stimulation. We have further utilized this assay to also examine cytokine expression in rabbit tissues during experimental syphilis infection.     

Processing of gene expression data generated by quantitative real-time RT-PCR

Quantitative real-time PCR represents a highly sensitive and powerful technique for the quantitation of nucleic acids. It has a tremendous potential for the high-throughput analysis of gene expression in research and routine diagnostics. However, the major hurdle is not the practical performance of the experiments themselves but rather the efficient evaluation and the mathematical and statistical analysis of the enormous amount of data gained by this technology, as these functions are not included in the software provided by the manufacturers of the detection systems. In this work, we focus on the mathematical evaluation and analysis of the data generated by quantitative real-time PCR, the calculation of the final results, the propagation of experimental variation of the measured values to the final results, and the statistical analysis. We developed a Microsoft Excel-based software application coded in Visual Basic for Applications, called Q-Gene, which addresses these points. Q-Gene manages and expedites the planning, performance, and evaluation of quantitative real-time PCR experiments, as well as the mathematical and statistical analysis, storage, and graphical presentation of the data. The Q-Gene software application is a tool to cope with complex quantitative real-time PCR experiments at a high-throughput scale and considerably expedites and rationalizes the experimental setup, data analysis, and data management while ensuring highest reproducibility.