荧光定量PCR技术研究进展

基于荧光能量传递技术的荧光定量PCR技术可实时监测PCR反应 ,准确敏感地测定模板浓度及检测基因变异等 ,它的出现 ,极大地克服了原有PCR技术存在的不足如交叉污染等问题 ,并改善了PCR技术的应用如可广泛应用于临床观测患者病情发展及预后 ,判断药物疗效等。本文概述了目前几种荧光定量PCR技术即Taqman、Amplisensor、分子信标、Lightcycler及复合探针法的原理及特点。便于更好地理解及应用这项技术。     

微滴数字PCR技术检测先天性心脏病患儿染色体22q11．2区段微缺失CSCD

目的探索一种检测22qii．2微缺失综合征的新方法。方法针对22q11．2微缺失综合征特异缺失区内的TBX1基因和内参基因RPP30设计引物和探针,采用微滴数字PCR（dropletdigitalPCR,ddPCR）的方法计算TBX1／RPP30的比值,检测22q11．2区段微缺失。结果通过数字PCR方法计算TBX1／尺PP30的比值检测22q11．2微缺失综合征,检出3例微阵列比较基因组杂交检测结果为22q11．2微缺失综合征阳性的样本。在14例临床诊断为先天性心脏病的患儿中检测出2例22q11．2微缺失阳性样本。结论微滴数字PCR可以准确检测出22q11．2区段微缺失,可提供一种快速、经济的检测先天性心脏病相关染色体22q11．2微缺失综合征的方法。     

数字PCR中微滴生成尺寸与频率的数值模拟

目的微滴合成是数字PCR中的关键技术,但其中两相流速与生成微滴大小和频率的关系尚不明确。本文采用VOF模型研究数字PCR系统中生成微滴的尺寸、频率与两相流速的关系。方法将氟化油作为连续相,反应液(水)作为离散相,通过求解整体的动量方程和各自相的体积分数连续方程来实现相与相间的界面追踪,模拟出微通道内两相的流动情况,对不同两相流速下微通道内微滴生成的尺寸和频率进行研究。结果在不同的流速条件下,微通道内会出现弹状流、滴状流和管状流3种流型。并且,对于弹状流和滴状流,随着连续相流速的增加,微液滴的生成尺寸减小,生成频率增加;而随着离散相流速的增加,微液滴的生成尺寸和频率都会增加。结论在滴状流状态下,当连续相流速为0.048~0.064 m/s,并且离散相流速为0.016~0.032 m/s时,可高效生成数字PCR微滴。     

实时荧光定量PCR的应用体会

对于病毒的监测和疾病的诊断,定性检测已不能满足临床需要,实时荧光定量PCR技术(realtime quantitative PCR)于1996年由美国Aplied Biosystems公司推出.     

实时定量PCR技术在病毒学研究中的应用

近年来发展起来的实时定量PCR技术与传统PCR技术相比,具有灵敏度和特异性高、重复性好、检测范围宽的优点,且极大降低了交叉污染的可能性。广泛应用于病毒感染的定量检测、临床疗效和药物评价、感染人群的普查以及感染源的监测、病毒与疾病之间关系的研究等方面。     

荧光定量PCR技术及其应用

荧光定量ＰＣＲ是新研制出的一种核酸定量技术。该技术在ＰＣＲ反应系统中引入了荧光标记探针,具有高灵敏性、高特异性和高精确性的特点。目前已被应用于病原体测定、肿瘤基因检测、基因表达、突变和多态性研究等多个领域。     

荧光定量 PCR与半定量PCR检测HBV DNA的对比分析

目的：对照分析荧光定量PCR与半定量PCR检测血清HBV DNA的载量，为临床基因检测实验室提供方法学选择依据。 方法： 取164份临床检测标本各用荧光定量PCR与半定量PCR检测，结果作统计学分析。 结果: 两种检测方法的灵敏度和特异度没有显著差异（χ2 =1.75，P>0.05）。 结论: 临床基因检测实验室可用半定量PCR法替代荧光定量 PCR法检测血清HBV DNA的载量。     

一种基于反相微乳液的数字PCR微滴的制备方法

目的提出一种新型的微滴生成方式——油包水(W/O)包裹技术,为数字PCR实验过程中微滴的制备提供一种更便捷的方式。方法通过配制一定比例的特定油相以及包含模板DNA等反应物的特定水相,在不同条件下形成W/O微滴,并进行PCR反应。结果所形成的微滴粒径大小均匀,并且在进行PCR反应过程中热力学性能保持不变,微滴不会因温度升降而破裂,适合于数字PCR中微滴的制备。结论生成的微滴可以有效地使模板DNA进行扩增反应。     

实时定量PCR技术在病毒学研究中的应用

近年来发展起来的实时定量PCR技术与传统PCR技术相比，具有灵敏度和特异性高、重复性好、检测范围宽的优点，且极大降低了交叉污染的可能性。广泛应用于病毒感染的定量检测、临床疗效和药物评价、感染人群的普查以及感染源的监测、病毒与疾病之间关系的研究等方面。     

利用荧光定量PCR技术对人肠道乳酸杆菌进行定量检测

目的应用荧光定量PCR技术对人粪便内乳酸杆菌进行定量检测,建立乳酸杆菌的荧光定量PCR检测体系。方法依据人肠道乳酸杆菌16S rDNA序列设计属特异性引物,应用荧光定量PCR技术检测乳酸杆菌的16S rDNA,对粪便中的乳酸杆菌进行定量检测和分析,并与用传统方法所获得的结果进行比较。结果荧光定量PCR检测乳酸杆菌和传统方法检测乳酸杆菌获得的结果接近,差异无统计学意义（P〉0.05）。结论荧光定量PCR技术比传统方法省时、省力,且敏感性和特异性更高。     

多体素1H磁共振频谱绝对定量脑内代谢物的方法

磁共振频谱(MRS)是一种利用磁共振原理和化学位移作用对一系列特定原子核及其化合物进行分析的方法.目前MRS的应用以1H谱最为广泛,其频谱技术已由单体素发展为三维多体素(3DCSI)扫描,为临床研究提供了许多重要的信息.就应用多体素磁共振频谱技术定量检测脑代谢物绝对浓度的方法进行综述.     

Digital PCR: a new technology for diagnosis of parasitic infections

BackgroundParasitic infections are responsible for a significant burden of disease worldwide as a result of international travel and immigration. More accurate diagnostic tools are necessary in support to parasite control and elimination programmes in endemic regions as well as for rapid case detection in non-endemic areas. Digital PCR (dPCR) is a powerful technology with recent applications in parasitology.AimsThis review provides for the first time an overview of dPCR as a novel technology applied to detection of parasitic infections, and highlights the most relevant potential benefits of this assay.SourcesPeer-reviewed literature pertinent to this review based on PubMed, Cochrane and Embase databases as well as laboratory experience of authors.ContentAmong the 86 studies retrieved, 17 used the dPCR applied to parasites belonging to protozoa (8), helminths (8) and arthropods (1) of clinical human interest. dPCR was adopted in four studies, respectively, for Plasmodium and Schistosoma japonicum. dPCR led to clear advantages over quantitative real-time PCR in P. falciparum and spp., and in S. japonicum showing higher sensitivity; and in Cryptosporidium with higher stability to inhibitors from stool. For all parasites, dPCR allows absolute quantitation without the need of a standard curve. Various dPCR platforms were used. A few critical factors need consideration: DNA load, choice of platform and reaction optimization.ImplicationsOwing to its sensitivity and quantitative characteristics, dPCR is a potential candidate to become an appealing new method among the molecular technologies for parasite detection and quantitative analysis in the future. In general, it has more applications than genomic DNA detection only, such as quantitation in mixed infections, gene expression and mutation analysis. dPCR should be considered in malaria screening and diagnosis as a complement to routine assays and in schistosomiasis elimination programmes. Standardized strategies and further studies are needed for the integration of dPCR in routine clinical laboratory.     

Detection of the copy number of plasmid encoding HBsAg in recombinant yeast by PCR relative quantitative assay

Thegeneticrecombinantyeast,Saccharomyces cerevisiae,carryingtheplasmidscontainingthe hepatitisBvirussurfaceantigen(HBsAg)encod ingregionisemployedtoproducetherecombinant hepatitisBvaccine.Someplasmidsoftherecom binantyeastmaybelostduringindustrialferment ationtherebyinfluencingtheexpressionofHB sAg.Eitheratthepracticalfermentationtoex pressHBsAgformakingvaccineorrenovationoffermentationprocessoftherecombinantyeastto enhancetheyieldsofHBsAg,suchasextension ofculturingperiod,theplasmidstat…     

Cellular chromosome DNA interferes with fluorescence quantitative real-time PCR detection of HBV DNA in culture medium

Fluorescence quantitative real-time PCR (FQ-PCR) is a recently developed technique increasingly used for clinical diagnosis by detection of hepatitis B virus (HBV) DNA in serum. FQ-PCR is also used in scientific research for detection of HBV DNA in cell culture. Understanding potential FQ-PCR interference factors can improve the accuracy of HBV DNA quantification in cell culture medium. HBV positive serum was diluted with culture medium to produce three test groups with HBV DNA levels of 5 x 10(7) copies/ml (high), 5 x 10(5) copies/ml (medium), and 5 x 10(3) copies/ml (low). Chromosome DNA was extracted from HepG2 cells and then added to high, medium, and low group samples at final concentrations of 0, 12.5, 25, 50, and 100 microg/ml. The samples were quantified by FQ-PCR and data were evaluated using statistical software. No marked changes were seen in the quantitative curves for high level HBV DNA samples when the samples were supplemented with 0-100 microg/ml of chromosome DNA. Interference was observed in medium level samples when 50 and 100 microg/ml of chromosome DNA was added. Interference was also observed in low level HBV DNA samples when the concentration of added chromosome DNA was greater than 25 microg/ml. The interference was eliminated when samples were digested by DNase I prior to PCR detection. In Conclusions, the presence of cellular chromosome DNA can interfere with the detection of HBV DNA by FQ-PCR. Removal of cellular chromosome DNA from culture media prior to FQ-PCR is necessary for reliable HBV DNA quantitative detection.     

Evaluation of reference genes and normalization strategy for quantitative real-time PCR in human pancreatic carcinoma

Histologically verified pairs (n=10) of pancreatic tumors and non-neoplastic tissues were used for quantitative real-time PCR and the stability of 24 reference genes was analyzed with geNorm and NormFinder software. Raw C{q} values correlated with the degree of RNA degradation. This correlation was abolished by normalization to C{q} of 18S endogenous control gene. Both geNorm and NormFinder programs suggested EIF2B1, ELF1, MRPL19, and POP4 as the same most stable genes. We have thus identified suitable reference genes for future expression studies in pancreatic carcinoma. Normalization method reducing the effects of RNA degradation on the quality of results was also developed.