A quantitative real time reverse-transcription polymerase chain reaction (qRT-PCR) assay with specific primers recommended by the World Health Organization (WHO) has been widely used successfully for detection and mon...A quantitative real time reverse-transcription polymerase chain reaction (qRT-PCR) assay with specific primers recommended by the World Health Organization (WHO) has been widely used successfully for detection and monitoring of the pandemic H1N1/2009 influenza A virus. In this study, we report the design and characterization of a novel set of primers to be used in a qRT-PCR assay for detecting the pandemic H1N1/2009 virus. The newly designed primers target three regions that are highly conserved among the hemagglutinin (HA) genes of the pandemic H1N1/2009 viruses and are different from those targeted by the WHO-recommended primers. The qRT-PCR assays with the newly designed primers are highly specific, and as specific as the WHO-recommended primers for detecting pandemic H1N1/2009 viruses and other influenza viruses including influenza B viruses and influenza A viruses of human, swine, and raccoon dog origin. Furthermore, the qRT-PCR assays with the newly designed primers appeared to be at least 10-fold more sensitive than those with the WHO-recommended primers as the detection limits of the assays with our primers and the WHO-recommended primers were 2.5 and 25 copies of target RNA per reaction, respectively. When tested with 83 clinical samples, 32 were detected to be positive using the qRT-PCR assays with our designed primers, while only 25 were positive by the assays with the WHO-recommended primers. These results suggest that the qRT-PCR system with the newly designed primers represent a highly sensitive assay for diagnosis of the pandemic H1N1/2009 virus infection.展开更多
Objective: To investigate molecular alterations associating with prostate carcinoma progression and potentially provide information toward more accurate prognosis/diagnosis. Methods: A set of laser captured microdisse...Objective: To investigate molecular alterations associating with prostate carcinoma progression and potentially provide information toward more accurate prognosis/diagnosis. Methods: A set of laser captured microdissected (LCM) speci- mens from 300 prostate cancer (PCa) patients undergoing radical prostatectomy (RP) were defined. Ten patients representing "aggressive" PCa, and 10 representing "non-aggressive" PCa were selected based on prostate-specific antigen (PSA) recurrence, Gleason score, pathological stage and tumor cell differentiation, with matched patient age and race between the two groups. Normal and neoplastic prostate epithelial cells were collected with LCM from frozen tissue slides obtained from the RP specimens. The expressions of a panel of genes, including NPY, PTEN, AR, AMACR, DD3, and GSTP1, were measured by quantitative real-time RT-PCR (TaqMan), and correlation was analyzed with clinicopathological features. Results: The expressions of AMACR and DD3 were consistently up-regulated in cancer cells compared to benign prostate epithelial cells in all PCa patients, whereas GSTP1 expression was down regulated in each patient. NPY, PTEN and AR exhibited a striking difference in their expression patterns between aggressive and non-aggressive PCas (P=0.0203, 0.0284, and 0.0378, respectively, Wilcoxon rank sum test). The lower expression of NPY showed association with "aggressive" PCas based on a larger PCa patient cohort analysis (P=0.0037, univariate generalized linear model (GLM) analysis). Conclusion: Despite widely noted heterogeneous nature of PCa, gene ex- pression alterations of AMACR, DD3, and GSTP1 in LCM-derived PCa epithelial cells suggest for common underlying mecha- nisms in the initiation of PCa. Lower NPY expression level is significantly associated with more aggressive clinical behavior of PCa; PTEN and AR may have potential in defining PCa with aggressive clinical behavior. Studies along these lines have potential to define PCa-associated gene expression alterations and likely co-regulation of genes/pathways critical in the biology of PCa onset/progression.展开更多
We isolated 4 Norwalk-like viruses (NLVs) contaminated oysters from 33 Chinese oysters collected from local commer- cial sources of Shandong Province. After amplification of the RNA-dependent RNA polymerase (RdRp) reg...We isolated 4 Norwalk-like viruses (NLVs) contaminated oysters from 33 Chinese oysters collected from local commer- cial sources of Shandong Province. After amplification of the RNA-dependent RNA polymerase (RdRp) region of NLVs genomes with RT-PCR, the open reading frame 1 (ORF1) of the RdRp was sequenced and subjected to multiple-sequence alignment. The re- sults showed that NLVs in the four isolates belong to genogroup II. The sequence comparison showed that the similarity between four Chinese oyster isolates were higher than 99.0%, which indicated that NLVs prevalent in close areas have high homogeneity in genome sequences. In addition, the most conserved sequences between diverse NLVs were used to design primers and TaqMan probes, then the real-time quantitative PCR assay was performed. According to the standard curve of GII NLVs, the original amounts (copies) of NLVs in positive patient’s fecal isolate, positive Japanese oyster isolate, and the Chinese oyster isolate were 8.9×108, 1.25×108 and 4.7×101 respectively. The detecting limit of NLVs was 1×101 copies. This study will be helpful for routine diagnosis of NLVs pathogens in foods and thus for avoiding food poisoning in the future.展开更多
在樱桃病毒A(CVA)mp基因保守区域设计了3对检测引物,经特异性筛选后,获得可用于病毒定量研究的引物。制备质粒标准品,建立标准曲线,同时验证该方法的灵敏度和特异性,并应用于田间果树样品CVA定量检测。最终成功筛选出1对检测效率高、特...在樱桃病毒A(CVA)mp基因保守区域设计了3对检测引物,经特异性筛选后,获得可用于病毒定量研究的引物。制备质粒标准品,建立标准曲线,同时验证该方法的灵敏度和特异性,并应用于田间果树样品CVA定量检测。最终成功筛选出1对检测效率高、特异性强的引物(CVA-dF2、CVA-dR2),基于SYBR Green I荧光染料建立反转录实时荧光定量PCR检测CVA的方法。该方法重复性好、灵敏度高,无需借助内参基因即可准确检测目的病毒载量,绝对定量标准曲线斜率为-3.5746,决定系数R2为0.9986,扩增效率为0.9044,比常规RT-PCR检测灵敏度高10倍。该方法的建立为CVA定量研究提供了有力工具,可用于果树中CVA批量检测或低丰度病毒样品检测。展开更多
基金supported by grants from National Basic Research Program of China (No.2011CB504800)National Natural Science Foundation of China (No. 31100128 and 81030031)+3 种基金National Mega Project on Major Drug Development (2009ZX09103-678)National Small Business Innovation and Research (SBIR) Program of Chinathe Technology R & D Program of Jiangsu Province, China (BG20077035 and BG2008662)NIH (RO1-AI041927,RO1-AI050468, RO1-DE014145, and RO1-DE014842)
文摘A quantitative real time reverse-transcription polymerase chain reaction (qRT-PCR) assay with specific primers recommended by the World Health Organization (WHO) has been widely used successfully for detection and monitoring of the pandemic H1N1/2009 influenza A virus. In this study, we report the design and characterization of a novel set of primers to be used in a qRT-PCR assay for detecting the pandemic H1N1/2009 virus. The newly designed primers target three regions that are highly conserved among the hemagglutinin (HA) genes of the pandemic H1N1/2009 viruses and are different from those targeted by the WHO-recommended primers. The qRT-PCR assays with the newly designed primers are highly specific, and as specific as the WHO-recommended primers for detecting pandemic H1N1/2009 viruses and other influenza viruses including influenza B viruses and influenza A viruses of human, swine, and raccoon dog origin. Furthermore, the qRT-PCR assays with the newly designed primers appeared to be at least 10-fold more sensitive than those with the WHO-recommended primers as the detection limits of the assays with our primers and the WHO-recommended primers were 2.5 and 25 copies of target RNA per reaction, respectively. When tested with 83 clinical samples, 32 were detected to be positive using the qRT-PCR assays with our designed primers, while only 25 were positive by the assays with the WHO-recommended primers. These results suggest that the qRT-PCR system with the newly designed primers represent a highly sensitive assay for diagnosis of the pandemic H1N1/2009 virus infection.
基金Project supported by the Center for Prostate Disease Researchthe Henry M. Jackson Foundation for the Advancement of Military Medicine, Rockville, MD, USA
文摘Objective: To investigate molecular alterations associating with prostate carcinoma progression and potentially provide information toward more accurate prognosis/diagnosis. Methods: A set of laser captured microdissected (LCM) speci- mens from 300 prostate cancer (PCa) patients undergoing radical prostatectomy (RP) were defined. Ten patients representing "aggressive" PCa, and 10 representing "non-aggressive" PCa were selected based on prostate-specific antigen (PSA) recurrence, Gleason score, pathological stage and tumor cell differentiation, with matched patient age and race between the two groups. Normal and neoplastic prostate epithelial cells were collected with LCM from frozen tissue slides obtained from the RP specimens. The expressions of a panel of genes, including NPY, PTEN, AR, AMACR, DD3, and GSTP1, were measured by quantitative real-time RT-PCR (TaqMan), and correlation was analyzed with clinicopathological features. Results: The expressions of AMACR and DD3 were consistently up-regulated in cancer cells compared to benign prostate epithelial cells in all PCa patients, whereas GSTP1 expression was down regulated in each patient. NPY, PTEN and AR exhibited a striking difference in their expression patterns between aggressive and non-aggressive PCas (P=0.0203, 0.0284, and 0.0378, respectively, Wilcoxon rank sum test). The lower expression of NPY showed association with "aggressive" PCas based on a larger PCa patient cohort analysis (P=0.0037, univariate generalized linear model (GLM) analysis). Conclusion: Despite widely noted heterogeneous nature of PCa, gene ex- pression alterations of AMACR, DD3, and GSTP1 in LCM-derived PCa epithelial cells suggest for common underlying mecha- nisms in the initiation of PCa. Lower NPY expression level is significantly associated with more aggressive clinical behavior of PCa; PTEN and AR may have potential in defining PCa with aggressive clinical behavior. Studies along these lines have potential to define PCa-associated gene expression alterations and likely co-regulation of genes/pathways critical in the biology of PCa onset/progression.
文摘We isolated 4 Norwalk-like viruses (NLVs) contaminated oysters from 33 Chinese oysters collected from local commer- cial sources of Shandong Province. After amplification of the RNA-dependent RNA polymerase (RdRp) region of NLVs genomes with RT-PCR, the open reading frame 1 (ORF1) of the RdRp was sequenced and subjected to multiple-sequence alignment. The re- sults showed that NLVs in the four isolates belong to genogroup II. The sequence comparison showed that the similarity between four Chinese oyster isolates were higher than 99.0%, which indicated that NLVs prevalent in close areas have high homogeneity in genome sequences. In addition, the most conserved sequences between diverse NLVs were used to design primers and TaqMan probes, then the real-time quantitative PCR assay was performed. According to the standard curve of GII NLVs, the original amounts (copies) of NLVs in positive patient’s fecal isolate, positive Japanese oyster isolate, and the Chinese oyster isolate were 8.9×108, 1.25×108 and 4.7×101 respectively. The detecting limit of NLVs was 1×101 copies. This study will be helpful for routine diagnosis of NLVs pathogens in foods and thus for avoiding food poisoning in the future.
文摘在樱桃病毒A(CVA)mp基因保守区域设计了3对检测引物,经特异性筛选后,获得可用于病毒定量研究的引物。制备质粒标准品,建立标准曲线,同时验证该方法的灵敏度和特异性,并应用于田间果树样品CVA定量检测。最终成功筛选出1对检测效率高、特异性强的引物(CVA-dF2、CVA-dR2),基于SYBR Green I荧光染料建立反转录实时荧光定量PCR检测CVA的方法。该方法重复性好、灵敏度高,无需借助内参基因即可准确检测目的病毒载量,绝对定量标准曲线斜率为-3.5746,决定系数R2为0.9986,扩增效率为0.9044,比常规RT-PCR检测灵敏度高10倍。该方法的建立为CVA定量研究提供了有力工具,可用于果树中CVA批量检测或低丰度病毒样品检测。
