一种利用微波炉加热快速提取细菌DNA用于PCR扩增的方法

利用微波加热原理,探索一种快速、简单、高效提取细菌DNA用于常规PCR快速检测的方法。在微波炉额定功率800 W条件下分别对大肠杆菌、金黄色葡萄球菌、单增李斯特氏菌、副溶血弧菌、沙门氏菌以及志贺氏菌6种常见食源性病原菌进行微波处理,离心取上清,获取细菌DNA,将样本DNA进行PCR扩增及琼脂糖凝胶电泳进行验证。并进一步采用微波加热方法针对6种病原菌最低检出浓度和混合提取6种病原菌DNA的PCR检测进行分析。6种病原菌在微波炉中加热40-130 s的上清DNA样本均能用于常规PCR扩增。针对检出浓度的分析表明,副溶血弧菌的最低检出浓度为103 cfu/ml,金黄色葡萄球菌为104 cfu/ml,肠出血性大肠杆菌为105 cfu/ml,肠炎沙门氏菌和福氏志贺氏菌的最低检出浓度为106 cfu/ml,针对单核细胞增生李斯特氏杆菌的的最低检出浓度为107 cfu/ml。进行6种混合菌微波共提取时,其中5种病原菌的特异性基因都能扩出,并且条带很亮。该微波提取方法具有快速,简单,高效的特点,能满足大部分食源性病原菌的常规PCR检测需求,大大节约了时间和成本,具有广泛的适用性,为细菌快速分子检测提供了简便手段。     

镍矿区紫茎泽兰根际及内生细菌多样性研究

利用变性梯度凝胶电泳(denaturing gradient gel electrophoresis, DGGE)技术对四川省攀枝花市镍矿区紫茎泽兰根际及不同器官中内生细菌多样性进行分析. 结果表明, 紫茎泽兰根际与不同器官内生细菌多样性存在明显差异, 香农 威纳指数(H)、丰度(S)及均匀度(EH)皆表现为根际土＞叶＞茎＞根. 土壤重金属含量与内生细菌多样性相关性分析表明, 样品的丰度(S)与Zn含量呈显著正相关 (P<0.05). 典型对应分析(cononical correspondence analysis, CCA)显示, 根际土细菌多样性受Zn、Cd、Pb、Ni等重金属负向影响; 紫茎泽兰叶内生细菌多样性与土壤Cu元素浓度呈负相关关系. DGGE条带回收测序结果显示, 根际及内生细菌主要为变形菌门(Proteobacteria)、异常球菌 栖热菌门(Deinococcus Thermus)、芽单胞菌门(Gemmatimonadetes)、放线菌门(Actinobacteria)及部分不可培养的细菌.     

牛肉及其制品中肉类掺假的荧光PCR鉴别体系优化

根据可能用于牛肉及其制品掺假的肉类原料(猪肉、鸡肉和鸭肉),设计了4对引物和探针.经过测试其具有良好的特异性后,对影响各自聚合酶链式反应(polymerase chain reaction,PCR)扩增的5个影响因素包括Mg2+浓度、Taq DNA聚合酶活、dNTPs浓度、引物和探针浓度以及模板DNA用量等进行了优化,确定了最佳的PCR扩增体系.同时,根据优化后的结果,对特异性进行进一步的测试,并使用建立的优化荧光PCR鉴别体系对混合样品和市售样品进行检测,确定整体检出限为0.1%(质量分数),并证明该鉴别体系的实际应用价值.     

应用PCR技术对DMD做基因诊断的研究

杜氏肌营养不良症，是致死性Ｘ链锁隐性遗传病。     

Real-time PCR的研究进展及其应用

本文综述了real-time PCR的原理、定量方法及其在各领域的应用。     

MGB probe assay for rapid detection of mtDNAl1778 mutation in the Chinese LHON patients by real-time PCR

Objective： Leber＇s hereditary optic neuropathy （LHON） is a maternally inherited degeneration of the optic nerve caused by point mutations of mitochondrial DNA （mtDNA）. Many unsolved questions regarding the penetrance and pathophysiological mechanism of LHON demand efficient and reliable mutation testing. This study aims to develop a minor groove binder （MGB） probe assay for rapid detection of mtDNA11778 mutation and heteroplasmy in Chinese LHON patients by real-time polymerase chain reaction （PCR）. Methods： Forty-eight patients suspected of having LHON and their maternal relatives underwent a molecular genetic evaluation, with 20 normal individuals as a control group at the same time. A real-time PCR involving two MGB probes was used to detect the mtDNA 1 1778 mutation and heteroplasmy. A linear standard curve was obtained by pUCmLHONG and pUCmLHONA clones. Results： All 48 LHON patients and their maternal relatives were positive for rntDNA11778 mutation in our assay, 27 heteroplasmic and 21 homoplasmic. Eighteen cases did not show an occurrence of the disease, while 9 developed the disease among the 27 heteroplasmic mutation cases. Eleven did not show an occurrence of the disease, while 10 cases developed the disease among 21 homoplasmic mutation cases. There was a significant difference in the incidence between the heteroplasmic and the homoplasmic mutation types. The time needed for running a real-time PCR assay was only 80 min. Conclusion： This real-time PCR assay is a rapid, reliable method for mtDNA mutation detection as well as heteroplasmy quantification. Detecting this ratio is very important for predicting phenotypic expression of unaffected carriers.     

Detection of genetically modified ingredients in seed samples from Heilongjiang soybean field

A total of 210 soybean samples collected from different areas of Heilongjiang Province were analyzed by PCR to detect the presence of RR (Roundup Ready) soybean ingredient. Results showed that CaMV35S promoter was not detected in all samples. CP4-EPSPS gene was amplified in 13 samples, but all of them were proved to be false positives in restriction endonuclease digestion analysis of PCR products. Further analysis by nested PCR indicated that there were no RR soybean ingredients in the samples. Besides, the amplified fragments of 0xl by CP4-EPSPS primers were sequenced, and the results confirmed again the fact that this fragment was not from CP4-EPSPS gene.     

在单细胞水平建立原位PCR基因鉴别技术平台——以精子基因型鉴别为例

目的精子发生是一个包括有丝分裂和减数分裂的精细调控过程,这一过程是从精原干细胞开始在雄性睾丸的曲细精管中完成的,由哺乳动物雌雄性比例接近1∶1这一现象可以推知:受精时带X基因的精子和带Y基因的精子比率应该是1∶1,但事实上精子发生的过程中,分化出来的X、Y精子的比例是否也为1∶1并不清楚,因为精子发生过程中并不是所有细胞都能最终形成精子,然而要研究雌雄出生比率就需要先研究精子本身是否严格地按照1∶1形成,因此本实验通过建立快速可靠原位PCR技术平台为进一步研究精子发生的调控过程提供支持。步骤用HSL基因优化荧光原位PCR实验方案,包括蛋白酶K作用时间和浓度,原位PCR前先对精子进行解聚,再用SRY基因对精子进行原位PCR鉴定。结果在荧光显微镜下可清楚地看到精子头部的荧光信号,头部有绿色荧光信号的精子为带有SRY基因的Y染色体精子(Y精子),实验检测到昆明小鼠精子共493个,有信号的共273个,X∶Y=55.37%,经卡方检验,X精子与Y精子的实验结果趋近于1∶1。结论可利用荧光原位PCR技术在单细胞水平快速鉴定精子"性别",理论上可用于任何细胞的任何基因的鉴定。     

传送流的软实时复用

MPEG2是由运动图像专家组织提出的图像及其伴音的通用编码标准,该标准在广电网络获得大量应用,MPEG2标准的研究是本论文的基础,论文对与本课题相关的MPEG2标准进行了说明,给出了软件实现传送流实时复用的方法,并详细介绍了其协议处理和时钟处理的原理和实现.最后对算法的性能和可行性进行了分析,得出实验结论.