Objective Establishing a highly sensitive real-time fluorescence quantitative PCR (qPCR) method for universal testing of epidemic African swine fever virus (ASFV) strains. Methods The ASFV p72 gene was targeted to des...Objective Establishing a highly sensitive real-time fluorescence quantitative PCR (qPCR) method for universal testing of epidemic African swine fever virus (ASFV) strains. Methods The ASFV p72 gene was targeted to design primer probes covering 24 p72 genotypes. The optimal amount of dimethylsulphoxide (DMSO) for qPCR amplification was determined, Various sensitivity and limit of detection (LOD) tests were performed, and clinical samples from China and imported goods were tested. Results The optimal primer-probe combination could specifically detect ASFV, 1.5% DMSO was optimal for qPCR, and LOD reached 3.2 copies/μL with good reproducibility (n = 20, p = 0.369). The method was employed to test 142 clinically suspected samples, of which 30 pig blood and 37 pig tissue samples were ASFV-positive. Moreover, the positive testing rate for ASFV was higher than for the standard qPCR method recommended by the Office International Des Epizooties (OIE), and for the commercially available kit. Thus, our method is superior for testing weakly positive samples with low virus titre, and epidemic strains present in imported goods. Conclusion Our method could be employed for universal testing of epidemic ASFV strains worldwide, ensuring wider coverage of hosts and ASFV strains/endemic strains, reducing false<span style="font-family:;" "=""> </span><span style="font-family:Verdana;">negatives, and benefitting early diagnosis.</span>展开更多
Recent advances in intelligent transportation system allow traffic safety studies to extend from historic data-based analyses to real-time applications. The study presents a new method to predict crash likelihood with...Recent advances in intelligent transportation system allow traffic safety studies to extend from historic data-based analyses to real-time applications. The study presents a new method to predict crash likelihood with traffic data collected by discrete loop detectors as well as the web-crawl weather data. Matched case-control method and support vector machines （SVMs） technique were employed to identify the risk status. The adaptive synthetic over-sampling technique was applied to solve the imbalanced dataset issues. Random forest technique was applied to select the contributing factors and avoid the over-fitting issues. The results indicate that the SVMs classifier could successfully classify 76.32% of the crashes on the test dataset and 87.52% of the crashes on the overall dataset, which were relatively satisfactory compared with the results of the previous studies. Compared with the SVMs classifier without the data, the SVMs classifier with the web-crawl weather data increased the crash prediction accuracy by 1.32% and decreased the false alarm rate by 1.72%, showing the potential value of the massive web weather data. Mean impact value method was employed to evaluate the variable effects, and the results are identical with the results of most of previous studies. The emerging technique based on the discrete traffic data and web weather data proves to be more applicable on real- time safety management on freeways.展开更多
Helicobacter pylori (H. pylori) is bacteria considered to be present in half of the population and it is a public health problem worldwide. Most patients infected with H. pylori show no clinical symptoms;nonetheless, ...Helicobacter pylori (H. pylori) is bacteria considered to be present in half of the population and it is a public health problem worldwide. Most patients infected with H. pylori show no clinical symptoms;nonetheless, approximately 10% to 20% of these patients will develop peptic ulcers and 1% will develop gastric cancer. The International Agency for Research on Cancer has classified H. pylori as a Group 1 carcinogen, recognized as the only bacteria capable of producing cancer. Samples of drinking water (n = 44) from aqueducts with chlorination treatment in selected areas with high prevalence of gastric cancer were analyzed in Costa Rica. Samples of drinking water from Panamá (n = 44) from aqueducts supplying untreated water for human consumption in the province of Chiriquí were also analyzed. The molecular marker of H. pylori, glmM, was used, and to optimize the Real Time PCR (qPCR) technique, annealing temperature, concentration of primers and probe were standardized;also, by analyzing different standard curves, the best reaction conditions that allowed detecting and quantifying the gene were determined. The LightCycler® 480 II (LC480II) equipment from Roche Diagnostics GmbH was used, as well as the Absolute Quantification Analysis by means of the Second Derivative Maximum Method. In the case of the samples from Costa Rica, it was determined that 79.5% were positive for H. pylori;removing outlier high average, quantification of bacteria was determined in 3.6 × 103 copies/100 mL. For Panamá it was determined that 86% of the samples were found positive for the presence of H. pylori;removing outlier high average quantification of bacteria was determined at 3.3 × 102 copies/100 mL. The difference in values between the aqueducts in both countries revealed an environmental distribution of the bacteria of epidemiological interest in each case.展开更多
Real time RT-qPCR(real-time quantitative reverse transcription-PCR)是一种快速、简便、准确、灵敏、成本低廉的基因检测技术,被认为是目前检测基因在转录水平表达的金标准。研究发现Real time RT-qPCR的检测结果会受到实验设计、...Real time RT-qPCR(real-time quantitative reverse transcription-PCR)是一种快速、简便、准确、灵敏、成本低廉的基因检测技术,被认为是目前检测基因在转录水平表达的金标准。研究发现Real time RT-qPCR的检测结果会受到实验设计、引物、模板的质量、内参的选择及数据分析的方法等多个因素的影响,规范实验设计及操作是得到可靠结论的前提和必要条件。对近年来国内外涉及Real time RT-qPCR整个实验流程及数据发表的一些规范及标准进行综述,以便规范实验设计及操作,加强实验质量控制,促进研究结果的推广,和更好地发挥其应用价值。展开更多
文摘Objective Establishing a highly sensitive real-time fluorescence quantitative PCR (qPCR) method for universal testing of epidemic African swine fever virus (ASFV) strains. Methods The ASFV p72 gene was targeted to design primer probes covering 24 p72 genotypes. The optimal amount of dimethylsulphoxide (DMSO) for qPCR amplification was determined, Various sensitivity and limit of detection (LOD) tests were performed, and clinical samples from China and imported goods were tested. Results The optimal primer-probe combination could specifically detect ASFV, 1.5% DMSO was optimal for qPCR, and LOD reached 3.2 copies/μL with good reproducibility (n = 20, p = 0.369). The method was employed to test 142 clinically suspected samples, of which 30 pig blood and 37 pig tissue samples were ASFV-positive. Moreover, the positive testing rate for ASFV was higher than for the standard qPCR method recommended by the Office International Des Epizooties (OIE), and for the commercially available kit. Thus, our method is superior for testing weakly positive samples with low virus titre, and epidemic strains present in imported goods. Conclusion Our method could be employed for universal testing of epidemic ASFV strains worldwide, ensuring wider coverage of hosts and ASFV strains/endemic strains, reducing false<span style="font-family:;" "=""> </span><span style="font-family:Verdana;">negatives, and benefitting early diagnosis.</span>
文摘目的本研究旨在建立一种实时荧光定量PCR方法,用于检测猕猴三磷酸腺苷结合盒转运蛋白G2(adenosine triphosphate-binding cassette transporter protein G2,ABCG2)mRNA的基因转录水平。方法使用NCBI上GenBank数据库猕猴(Macaca mulatta)的ABCG2核苷酸序列号NM_001032919.1及内参GAPDH核苷酸序列号NM_001195426.1,借助Primer premier 5.0软件设计PCR引物。提取猕猴新鲜肾组织的总RNA,并反转录合成cDNA。接着,利用PCR引物进行实时荧光定量PCR扩增,并根据反应体系中荧光的变化情况定量分析ABCG2的mRNA相对表达水平。结果PCR产物测序结果显示,扩增的ABCG2和GAPDH核苷酸序列与NCBI上猕猴的序列同源性分别为90.91%和91.14%。ABCG2和GAPDH的扩增效率均达到80%~120%,实时荧光定量PCR标准曲线的熔解曲线为单峰,R2接近1。结论本研究建立的检测猕猴ABCG2 mRNA实时荧光定量检测方法,为研究高尿酸血症的发病机制以及新药开发奠定基础。
基金supported by the National Natural Science Foundation (71301119)the Shanghai Natural Science Foundation (12ZR1434100)
文摘Recent advances in intelligent transportation system allow traffic safety studies to extend from historic data-based analyses to real-time applications. The study presents a new method to predict crash likelihood with traffic data collected by discrete loop detectors as well as the web-crawl weather data. Matched case-control method and support vector machines （SVMs） technique were employed to identify the risk status. The adaptive synthetic over-sampling technique was applied to solve the imbalanced dataset issues. Random forest technique was applied to select the contributing factors and avoid the over-fitting issues. The results indicate that the SVMs classifier could successfully classify 76.32% of the crashes on the test dataset and 87.52% of the crashes on the overall dataset, which were relatively satisfactory compared with the results of the previous studies. Compared with the SVMs classifier without the data, the SVMs classifier with the web-crawl weather data increased the crash prediction accuracy by 1.32% and decreased the false alarm rate by 1.72%, showing the potential value of the massive web weather data. Mean impact value method was employed to evaluate the variable effects, and the results are identical with the results of most of previous studies. The emerging technique based on the discrete traffic data and web weather data proves to be more applicable on real- time safety management on freeways.
文摘Helicobacter pylori (H. pylori) is bacteria considered to be present in half of the population and it is a public health problem worldwide. Most patients infected with H. pylori show no clinical symptoms;nonetheless, approximately 10% to 20% of these patients will develop peptic ulcers and 1% will develop gastric cancer. The International Agency for Research on Cancer has classified H. pylori as a Group 1 carcinogen, recognized as the only bacteria capable of producing cancer. Samples of drinking water (n = 44) from aqueducts with chlorination treatment in selected areas with high prevalence of gastric cancer were analyzed in Costa Rica. Samples of drinking water from Panamá (n = 44) from aqueducts supplying untreated water for human consumption in the province of Chiriquí were also analyzed. The molecular marker of H. pylori, glmM, was used, and to optimize the Real Time PCR (qPCR) technique, annealing temperature, concentration of primers and probe were standardized;also, by analyzing different standard curves, the best reaction conditions that allowed detecting and quantifying the gene were determined. The LightCycler® 480 II (LC480II) equipment from Roche Diagnostics GmbH was used, as well as the Absolute Quantification Analysis by means of the Second Derivative Maximum Method. In the case of the samples from Costa Rica, it was determined that 79.5% were positive for H. pylori;removing outlier high average, quantification of bacteria was determined in 3.6 × 103 copies/100 mL. For Panamá it was determined that 86% of the samples were found positive for the presence of H. pylori;removing outlier high average quantification of bacteria was determined at 3.3 × 102 copies/100 mL. The difference in values between the aqueducts in both countries revealed an environmental distribution of the bacteria of epidemiological interest in each case.
文摘Real time RT-qPCR(real-time quantitative reverse transcription-PCR)是一种快速、简便、准确、灵敏、成本低廉的基因检测技术,被认为是目前检测基因在转录水平表达的金标准。研究发现Real time RT-qPCR的检测结果会受到实验设计、引物、模板的质量、内参的选择及数据分析的方法等多个因素的影响,规范实验设计及操作是得到可靠结论的前提和必要条件。对近年来国内外涉及Real time RT-qPCR整个实验流程及数据发表的一些规范及标准进行综述,以便规范实验设计及操作,加强实验质量控制,促进研究结果的推广,和更好地发挥其应用价值。
