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《微生物学免疫学进展》2016,(2)
目的对地高辛标记DNA探针杂交法检测人用狂犬病疫苗(Vero细胞)DNA残留量进行适用性验证及应用。方法对地高辛标记DNA探针杂交法检测人用狂犬病疫苗(Vero细胞)DNA残留量进行特异性、灵敏度及稳定性验证,并应用该方法检测3批人用狂犬病疫苗(Vero细胞)的DNA残留量。结果地高辛标记探针的标记效率为0.1 pg。验证结果显示探针与非同源DNA无杂交;最低检测限度为1 pg;探针在-20℃放置7个月后,检测灵敏度仍可达到1 pg;3批人用狂犬病疫苗(Vero细胞)中残留DNA含量均符合《中国药典》规定质量控制标准。结论地高辛标记DNA探针杂交法特异性、灵敏度好,结果稳定,适用于人用狂犬病疫苗(Vero细胞)中Vero细胞DNA残留量的检测及疫苗生产过程和其成品的质量控制,对其他以Vero细胞为基质的病毒性疫苗质量控制具有借鉴意义。 相似文献
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目的:应用定量荧光原位杂交(Q-FISH)方法测定端粒长度。方法:选取4种端粒长度均一的标准细胞株采用Q-FISH的方法做出荧光亮度与端粒长度的标准曲线,从而得出实验细胞株的端粒长度,与DNA印迹法测定末端限制性片段(TRF)长度进行二者之间的相关性分析。结果:检测荧光强度的最佳线性曝光时间为400ms,相对于DNA印迹法,定量荧光原位杂交(Q-FISH)法所需标本量少,实验周期短,端粒长度结果与Southern杂交法具有很好的相关性。结论:采用定量荧光原位杂交方法测端粒长度具有重复性好、精确可靠的特点,适用于对珍贵标本的端粒改变进行分析。 相似文献
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目的:端粒是真核生物染色体末端的一种高度保守的负责维持染色体稳定的特殊结构,其DNA序列长度即端粒长度,会随着年龄增长或疾病发生发展而逐渐缩短,检测端粒长度可以为评估机体衰老和健康状况提供参考,但目前缺乏测定微量牛DNA样本绝对端粒长度的方法;通过实时荧光定量PCR(real-time quantitative PCR, qPCR)实现微量牛DNA样本绝对端粒长度的测定并评估DNA提取方法对牛绝对端粒长度测定结果的影响,为进行端粒长度研究时选择合适的DNA提取方法和端粒长度分析方法提供参考。方法:利用标准曲线对检测样本的端粒和内参Ct值进行转换,通过qPCR测定牛端粒长度绝对值;采用膜吸附法、苯酚-氯仿法和磁珠法3种方法分别提取相同样本的DNA,分别用端粒末端限制性片段(terminal restriction fragment, TRF)分析法和qPCR法分析端粒长度,比较不同DNA提取方法对牛绝对端粒长度测定的影响。结果:(1)qPCR可以测定纳克级别DNA样本的绝对端粒长度,检测结果重复性良好,并且和“金标准”TRF测定结果的相关性良好。(2)不同方法提取的DNA用TRF分析法和... 相似文献
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目的建立利用流式荧光原位杂交法检测细胞端粒长度的技术方法。方法以端粒酶敲除的G3小鼠和同龄野生型小鼠为检测对象,分离其外周血中的单个核细胞后与肽核酸荧光探针杂交,用流式细胞仪采集和分析其端粒长度,分别用荧光原位杂交方法和SYBR Green荧光定量PCR方法验证其准确性。结果流式荧光原位杂交法测定G3小鼠细胞端粒相对长度与C57BJ/6野生型小鼠相比为0.5345,荧光定量PCR测定端粒相对长度为0.5717,结果基本一致。结论流式细胞术与原位杂交方法结合起来检测细胞端粒的平均长度可靠易行,对单个核细胞端粒平均长度的检测有较高的实用性。 相似文献
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目的:建立化学发光法检测体外HBV复制水平的方法,研究其灵敏度和稳定性.方法:用HBV DNA重组质粒pCH9转染到人肝癌细胞株HepG2和Huh7中,5d后收集细胞并抽提其HBV复制中问体DNA,转印后以地高辛标记HBV DNA为探针进行杂交,用化学发光法检测杂交结果,同时进行探针灵敏度的检测.结果:转染后HepG2和Huh7细胞提取HBV DNA中检测出较强的复制中间体的信号,分别为松散环状DNA(rcDNA),双链线性DNA(dslDNA),单链DNA(ssDNA),探针检测的灵敏度可达到lpg,接近同位素法检测的灵敏度.整个实验重复3次获得同样结果.结论:成功建立了稳定的化学发光法检测体外HBV复制水平的方法. 相似文献
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目的:研究腹透液相关浓度葡萄糖(1.5%、2.5%)对人腹膜间皮细胞端粒长度的影响及葛根素的拮抗作用.方法:人腹膜间皮细胞在含糖(1.5%、2.5%)、含糖含葛根素(葛根素终浓度为100μg/ml)的无血清培养基培养24h后,收集细胞,提取DNA,采用Southerm杂交测定细胞端粒长度.结果:经含糖1.5%和2.5%培养基培养24h后腹膜间皮细胞大多出现肥大.高浓度葡萄糖组细胞端粒长度缩短,2.5%组和1.5%组无显著差别(P>0.05).葛根素加入后含糖组腹膜间皮细胞端粒长度延长(P<0.05).结论:高糖可使间皮细胞端粒缩短,且可能与高糖作用下细胞肥大有关,葛根素可拮抗高糖对间皮细胞端粒长度的影响. 相似文献
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[目的]随着高通量测序方法的应用,越来越多的sRNA (small non-coding RNA,sRNA)需验证.本研究建立用地高辛标记Northern blot检测鼠疫菌sRNA的技术,为细菌sRNA验证提供一种灵敏、特异的方法.[方法]在低铁条件下,提取鼠疫菌总RNA,10% dPAGE分离后电转到尼龙膜上并用紫外线交联RNA.膜经地高辛标记RyhB1或RyhB2寡核苷酸RNA探针过夜杂交后洗脱、封闭和免疫检测,最后曝光显影.[结果]地高辛标记的Northern blot曝光时间为20 s-3 min,RyhB1或RyhB2检测灵敏度分别为0.005 μg和0.05 μg.RyhB1或RyhB2探针特异性好,相互间无交叉反应.带正电或中性的尼龙膜都适用于杂交反应.RNA探针在42℃-65℃内杂交均可,提高温度可减少非特异性反应;而DNA探针杂交温度需摸索.[结论]本研究成功构建一种地高辛标记Northern blot检测鼠疫菌sRNA技术,具有特异性好、灵敏度高、探针易保存、曝光时间短等优点,为细菌sRNA验证和功能研究提供有利工具. 相似文献
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自从Southern(1975)首次进行DNA探针杂交后,至今核酸分子杂交已成为分子生物学的最基本方法。Matthews和Kricka[1]总结了各种杂交方法,将其归为两大类:一是异相杂交(heterogeneousassay)即固相杂交,目的核酸结合于不溶性支持物上;二是同相杂交(homogeneousassay)即液相杂交,一般同时使用两个探针。为了检测杂交,寡核苷酸探针需要标记,探针的标记物有放射性同位素和非放射性标记物。固相杂交常使用放射性同位素,荧光素是一种非放射性标记物,它能检测到的DNA浓度比吸收减色测定方法所需DNA浓度低100-1000倍[2],在同相杂交中广泛用于探针的标记。最近,荧光探针研究获得了新的进展,Tyagi和Krammer(1996)建立了一种新的荧光探针-分子信标探针,并得到许多应用,我们实验室也开展了这方面的研究。本文拟对荧光探针的研究进展作一综述。 相似文献
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Longitudinal studies of human leucocyte telomere length often report a percentage of individuals whose telomeres appear to lengthen. However, based on theoretical considerations and empirical data, Steenstrup et al. (Nucleic Acids Research, 2013, vol 41(13): e131) concluded that this lengthening is unlikely to be a real biological phenomenon and is more likely to be an artefact of measurement error. We dispute the logic underlying this claim. We argue that Steenstrup et al.'s analysis is incomplete because it failed to compare predictions derived from assuming a scenario with no true telomere lengthening with alternative scenarios in which true lengthening occurs. To address this deficit, we built a computational model of telomere dynamics that allowed us to compare the predicted percentage of observed telomere length gainers given differing assumptions about measurement error and the true underling dynamics. We modelled a set of scenarios, all assuming measurement error, but both with and without true telomere lengthening. We found a range of scenarios assuming some true telomere lengthening that yielded either similar or better quantitative fits to the empirical data on the percentage of individuals showing apparent telomere lengthening. We conclude that although measurement error contributes to the prevalence of apparent telomere lengthening, Steenstrup et al.'s conclusion was too strong, and current data do not allow us to reject the hypothesis that true telomere lengthening is a real biological phenomenon in epidemiological studies. Our analyses highlight the need for process‐level models in the analysis of telomere dynamics. 相似文献
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端粒随细胞分裂进行性缩短不但防止了人类肿瘤的发展,而且与人类的衰老密切相关。另外,端粒中存在一种特殊的现象:端粒位置效应,它首先在酵母中发现,表现为靠近端粒序列附近的基因表达因端粒的位置效应而沉默。在人类细胞中也存在端粒位置效应,并且有多种因子参与此效应,它可能对细胞生长停止、肿瘤以及衰老发生时等许多随端粒缩短密切相关基因的程序性表达产生重要作用。 相似文献
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Telomeres often shorten with time, although this varies between tissues, individuals and species, and their length and/or rate of change may reflect fitness and rate of senescence. Measurement of telomeres is increasingly important to ecologists, yet the relative merits of different methods for estimating telomere length are not clear. In particular the extent to which interstitial telomere sequences (ITSs), telomere repeats located away from chromosomes ends, confound estimates of telomere length is unknown. Here we present a method to estimate the extent of ITS within a species and variation among individuals. We estimated the extent of ITS by comparing the amount of label hybridized to in‐gel telomere restriction fragments (TRF) before and after the TRFs were denatured. This protocol produced robust and repeatable estimates of the extent of ITS in birds. In five species, the amount of ITS was substantial, ranging from 15% to 40% of total telomeric sequence DNA. In addition, the amount of ITS can vary significantly among individuals within a species. Including ITSs in telomere length calculations always underestimated telomere length because most ITSs are shorter than most telomeres. The magnitude of that error varies with telomere length and is larger for longer telomeres. Estimating telomere length using methods that incorporate ITSs, such as Southern blot TRF and quantitative PCR analyses reduces an investigator's power to detect difference in telomere dynamics between individuals or over time within an individual. 相似文献
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Restriction Enzyme-Resistant High Molecular Weight Telomeric DNA Fragments in Tobacco 总被引:4,自引:0,他引:4
Restriction endonuclease-resistant high-molecular-weight (HMW)DNA fragments were isolated from nuclear DNA fragments in tobacco.The size of the fragments produced by EcoRI, HindIII, AfaI,and HaeIII ranged from 20 kb to over 166 kb. The kinetics ofdigestion by Bal31 nuclease showed that most of the HMW fragmentsare chromosome ends. The consensus sequence for tobacco telomererepeats was determined to be CCCTAAA by genomic sequencing usingthe HMW fragments and by sequencing after cloning. Besides thetelomere sequence, 9 tandem repeats of a 45-bp sequence wereidentified, in which a 35-bp unit sequence (AGTCAGCATTAGGGTTTTAAACCCTAAACTGAACT)formed a stem structure. The front of the stem is composed ofa palindrome of the telomere repeats. This highly conservedunit is surrounded by less conserved internal sequences thatare around 1011 bp in size and contain a TTTT stretch.The internal sequences resemble the 1011 bp consensusfor the scaffold attachment regions found in yeast and drosophila.The characteristic 45-bp sequence was abundant on the ends ofchromosomes. The shortest distance between the repeats containingtelomeric stem and the telomere was less than 20 kb. This architectureof the tobacco chromosome end region resembles the end regionof yeast chromosomes in which autonomous replication sequencesare present frequently. 相似文献