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
目的以微小RNA(miRNA)作为检测样本,考察影响实时荧光定量聚合酶链反应(RT-qPCR)检测结果的关键点,并筛选出最优的检测方法。方法采用3种miRNA提取方法(分别使用EasyPure^(■)miRNA提取试剂盒、miRNA提取试剂盒、TransZol Up Plus RNA...目的以微小RNA(miRNA)作为检测样本,考察影响实时荧光定量聚合酶链反应(RT-qPCR)检测结果的关键点,并筛选出最优的检测方法。方法采用3种miRNA提取方法(分别使用EasyPure^(■)miRNA提取试剂盒、miRNA提取试剂盒、TransZol Up Plus RNA提取试剂盒)和3种反转录方法(分别使用TransScript^(■)第一链cDNA反转录试剂盒、Evo M-MLV反转录试剂盒、miRNA第一链cDNA合成试剂盒)处理大鼠纹状体脑区,检测miRNA与c DNA的质量和效率,并使用常规RT-qPCR检测miRNA水平,比较不同方法所得结果。结果3种RNA提取方法得到的miRNA质量和效率比较差异均有统计学意义,其中方法2的效果较好,但方法1、2、3的RT-qPCR结果比较差异无统计学意义(miR-132:25.91±9.79、25.26±10.25、27.28±7.39,miR-U6:27.98±11.25、25.98±9.78、29.62±9.65,均P>0.05);3种反转录方法所得实验结果比较差异有统计学意义,方法3所得结果明显低于方法1、方法2(miR-132:16.53±3.17比35.20±1.06、31.42±2.95,miR-U6:16.63±1.73比36.06±2.57、35.59±1.54,均P<0.05),主要影响RT-qPCR的扩增效率和扩增特异性。结论使用能高效富集约18 nt大小RNA的提取方法和在miRNA的3’末端加多聚A尾(Poly A)的反转录酶,可以得到更可靠的RT-qPCR结果。展开更多
目的探讨骨桥蛋白(OPN)mRNA在口腔鳞状细胞癌(OSCC)及配对正常口腔黏膜组织中的表达及临床意义。方法用SYBR Green I荧光定量RT-PCR方法检测30例OSCC患者肿瘤组织及其配对的正常口腔粘膜中OPN mRNA的表达。结果Real time RT-PCR检测结...目的探讨骨桥蛋白(OPN)mRNA在口腔鳞状细胞癌(OSCC)及配对正常口腔黏膜组织中的表达及临床意义。方法用SYBR Green I荧光定量RT-PCR方法检测30例OSCC患者肿瘤组织及其配对的正常口腔粘膜中OPN mRNA的表达。结果Real time RT-PCR检测结果表明,OPN mRNA在OSCC和配对正常粘膜的表达水平分别为4.17±0.51和0.97±0.12,上调4.3倍(P<0.001)。在30例OSCC标本中,OPN mRNA在高分化鳞癌中的表达为2.16±0.17,中/低分化鳞状细胞癌中的表达为6.80±0.72,下调3.1倍,P<0.05;在颈淋巴结阳性组表达为6.38±0.56,颈淋巴结阴性组为2.89±0.32,上调2.2倍,P<0.05;在临床早期表达为2.34±0.17,临床晚期表达为4.73±0.35,下调2.0倍,P<0.05。结论在OSCC的癌变过程中OPN基因的表达水平显著上调,可能是OSCC诊断、治疗及预后判断的一个靶点基因;OPN在OSCC分类诊断中有应用价值。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.
文摘目的以微小RNA(miRNA)作为检测样本,考察影响实时荧光定量聚合酶链反应(RT-qPCR)检测结果的关键点,并筛选出最优的检测方法。方法采用3种miRNA提取方法(分别使用EasyPure^(■)miRNA提取试剂盒、miRNA提取试剂盒、TransZol Up Plus RNA提取试剂盒)和3种反转录方法(分别使用TransScript^(■)第一链cDNA反转录试剂盒、Evo M-MLV反转录试剂盒、miRNA第一链cDNA合成试剂盒)处理大鼠纹状体脑区,检测miRNA与c DNA的质量和效率,并使用常规RT-qPCR检测miRNA水平,比较不同方法所得结果。结果3种RNA提取方法得到的miRNA质量和效率比较差异均有统计学意义,其中方法2的效果较好,但方法1、2、3的RT-qPCR结果比较差异无统计学意义(miR-132:25.91±9.79、25.26±10.25、27.28±7.39,miR-U6:27.98±11.25、25.98±9.78、29.62±9.65,均P>0.05);3种反转录方法所得实验结果比较差异有统计学意义,方法3所得结果明显低于方法1、方法2(miR-132:16.53±3.17比35.20±1.06、31.42±2.95,miR-U6:16.63±1.73比36.06±2.57、35.59±1.54,均P<0.05),主要影响RT-qPCR的扩增效率和扩增特异性。结论使用能高效富集约18 nt大小RNA的提取方法和在miRNA的3’末端加多聚A尾(Poly A)的反转录酶,可以得到更可靠的RT-qPCR结果。
文摘目的探讨骨桥蛋白(OPN)mRNA在口腔鳞状细胞癌(OSCC)及配对正常口腔黏膜组织中的表达及临床意义。方法用SYBR Green I荧光定量RT-PCR方法检测30例OSCC患者肿瘤组织及其配对的正常口腔粘膜中OPN mRNA的表达。结果Real time RT-PCR检测结果表明,OPN mRNA在OSCC和配对正常粘膜的表达水平分别为4.17±0.51和0.97±0.12,上调4.3倍(P<0.001)。在30例OSCC标本中,OPN mRNA在高分化鳞癌中的表达为2.16±0.17,中/低分化鳞状细胞癌中的表达为6.80±0.72,下调3.1倍,P<0.05;在颈淋巴结阳性组表达为6.38±0.56,颈淋巴结阴性组为2.89±0.32,上调2.2倍,P<0.05;在临床早期表达为2.34±0.17,临床晚期表达为4.73±0.35,下调2.0倍,P<0.05。结论在OSCC的癌变过程中OPN基因的表达水平显著上调,可能是OSCC诊断、治疗及预后判断的一个靶点基因;OPN在OSCC分类诊断中有应用价值。
