Real-time PCR is a closed DNA amplification system that skillfully integrates biochemical, photoelectric and computer techniques. Fluorescence data acquired once per cycle provides rapid absolute quantification of ini...Real-time PCR is a closed DNA amplification system that skillfully integrates biochemical, photoelectric and computer techniques. Fluorescence data acquired once per cycle provides rapid absolute quantification of initial template copy numbers as PCR products are generated. This technique significantly simplifies and accelerates the process of producing reproducible quantification of nucleic acid molecules. It not only is a sensitive, accurate and rapid quantitative method, but it also provides an easier way to calculate the absolute starting copy number of nucleic acid molecules to be tested. Together with molecular bio-techniques, like microarray, real-time PCR will play a very important role in many aspects of molecular life science such as functional gene analysis and disease molecular diagnostics. This review introduces the detailed principles and application of the real-time PCR technique, describes a recently developed system for exact quantification of AUX/IAA genes In Arabidopsis, and discusses the problems with the real-time PCR process.展开更多
针对数字液滴聚合酶链式反应(droplet digital Polymerase Chain Reaction,ddPCR)核酸检测中数字聚合酶链式反应由液滴数量多、尺寸小、排列紧密、荧光强度不均匀而导致的液滴难以计数问题,提出一种图像处理方法,基于灰度遍历法采集图...针对数字液滴聚合酶链式反应(droplet digital Polymerase Chain Reaction,ddPCR)核酸检测中数字聚合酶链式反应由液滴数量多、尺寸小、排列紧密、荧光强度不均匀而导致的液滴难以计数问题,提出一种图像处理方法,基于灰度遍历法采集图像的信息,通过微分分析法对液滴进行分类和计数,可准确采集ddPCR实验的图像信息。通过卷积算法去除图像中的噪声,使用灰度分布均衡化法增强图像的对比度。以灰度遍历的方式将图像在逐个阈值下二值化,并以几何条件为限制统计液滴数量。通过微分分析法对数据进行分析,得出荧光液滴与全部液滴的计数结果。在以人类gDNA(genomic DNA)为检测样本的ddPCR实验中,该算法的平均检测准确率为99.36%,与商用仪器算法和同类算法相比分别提高了2.24%,2.53%。该方法为ddPCR实验提供了可靠的检测结果,可更好地适用于ddPCR实验。展开更多
Using primers complementary to the conserved sequence previously published of BTV genomic dsRNA segment 7,a part of the 5′end of segment 7 was synthesized and amplified by RT PCR method.We adapted this method to test...Using primers complementary to the conserved sequence previously published of BTV genomic dsRNA segment 7,a part of the 5′end of segment 7 was synthesized and amplified by RT PCR method.We adapted this method to test on 12 blood samples suspected to be with BTV and the blood sample of a health sheep as a negative control.The results suggested that eleven of the twelve samples were positive,one was negative.In order to compare the sensitivity and reliability of RT PCR technique,Vero cells were used to isolate BTV from these suspected samples and the results will be a control.The study proved that RT PCR is a rapid,sensitive and precise method for detection and identification of bluetongue virus in clinical samples,animal quarantine and scientific research.展开更多
文摘Real-time PCR is a closed DNA amplification system that skillfully integrates biochemical, photoelectric and computer techniques. Fluorescence data acquired once per cycle provides rapid absolute quantification of initial template copy numbers as PCR products are generated. This technique significantly simplifies and accelerates the process of producing reproducible quantification of nucleic acid molecules. It not only is a sensitive, accurate and rapid quantitative method, but it also provides an easier way to calculate the absolute starting copy number of nucleic acid molecules to be tested. Together with molecular bio-techniques, like microarray, real-time PCR will play a very important role in many aspects of molecular life science such as functional gene analysis and disease molecular diagnostics. This review introduces the detailed principles and application of the real-time PCR technique, describes a recently developed system for exact quantification of AUX/IAA genes In Arabidopsis, and discusses the problems with the real-time PCR process.
文摘Using primers complementary to the conserved sequence previously published of BTV genomic dsRNA segment 7,a part of the 5′end of segment 7 was synthesized and amplified by RT PCR method.We adapted this method to test on 12 blood samples suspected to be with BTV and the blood sample of a health sheep as a negative control.The results suggested that eleven of the twelve samples were positive,one was negative.In order to compare the sensitivity and reliability of RT PCR technique,Vero cells were used to isolate BTV from these suspected samples and the results will be a control.The study proved that RT PCR is a rapid,sensitive and precise method for detection and identification of bluetongue virus in clinical samples,animal quarantine and scientific research.
