共查询到17条相似文献，搜索用时 326 毫秒
1.
目的应用PCR-DGGE（denaturing gradient gel electrophoresis,变性梯度凝胶电泳）技术对孤独症家庭成员肠道微生物菌群结构进行研究.材料和方法提取粪便细菌总基因组DNA,PCR扩增细菌16S rDNA基因V3可变区,DGGE方法检测PCR产物.结果建立了孤独症家庭成员肠道菌群组成的DGGE指纹图谱,分析结果显示孤独症儿童肠道菌群结构的相似度为0.42-0.65,家庭内孤独症儿童与父母之间的相似度为0.42-0.68.结论 PCR-DGGE技术是一种快速有效的用于分析研究人体肠道菌群结构的技术.孤独症儿童肠道菌群结构与父母的相似度较高,提示生活环境对肠道菌群的组成有影响. 相似文献
2.
酸奶在储存过程中菌群结构变化研究 总被引：1，自引：0，他引：1
张红琳 《南京晓庄学院学报》2010,26(6):44-46
为了探究酸奶储存过程中微生物菌群结构的变化,为其长期贮存奠定理论基础.采用酚/氯仿法抽提细菌基因组DNA,以此作为PCR反应的模板,进行16S rDNA基因有效扩增,并将PCR扩增产物进行DGGE电泳.用Bionumerics软件对DGGE分子指纹图谱进行舌苔菌群结构相似性分析.实验结果表明,采用该方法成功地扩增出16S rDNA V3区片段,为230 bp.DGGE分子指纹图谱结果表明,1、2号酸奶样品高相似性为93%,1-3号与4-7号的相似仅为6%,表明酸奶贮存过程中,菌群结构发生了变化. 相似文献
3.
采用玻璃珠法从高温堆肥样品中进行了微生物总DNA的提取,以细菌16S rRNA基因 V3区通用引物进行了PCR扩增,随后用变性梯度凝胶电泳(DGGE)技术对其微生物多样性进行评估.结果表明,玻璃珠法能够快速、有效地获得高质量的堆肥微生物总DNA,所得的DNA分子片段在15kb以上,使用细菌16S rRNA 基因通用引物(27F和1492R)对总DNA进行PCR扩增,获得了近全长的16S rDNA序列(约1.5kb);DGGE分析结果表明,用该方法提取到的DNA种类较为丰富,多样性较好,能够进一步应用于群落结构分析.因此,玻璃珠法提取的DNA可以用于堆肥微生物的分子生态学研究及微生物群落结构分析. 相似文献
4.
探究烤鸭储存过程中菌群的变化,为延长其贮存期奠定理论基础．用酚、氯仿、异戊醇法提取烤鸭肉中细菌的基因组DNA,PCR扩增16SRNAV3区,用DGGE电泳技术分析菌群结构的变化情况,然后用Bionumerics软件对DGGE分子指纹图谱进行烤鸭肉菌群结构相似性分析．PCR结果表明,成功扩增了细菌基因组DNA,为230bp;DGGE实验结果表明,DGGE分子指纹图谱上条带存在显著差异;用bionumberics软件分析,发现样本相似度不断变化,表明在储存过程中,烤鸭肉菌群结构发生了变化．该研究为延长烤鸭肉保质期提供理论参考依据． 相似文献
5.
研究禽肉储存过程中菌群的变化,为其长期贮存奠定理论基础.采用酚氯仿异戊醇法提取禽肉中菌群基因组DNA,并通过PCR扩增及DGGE电泳技术探讨其随保存时间延长,菌群结构变化情况.结果表明,成功扩增了菌群基因组DNA,为230bp.DGGE电泳结果发现,DGGE分子指纹图谱上条带存在显著的差异,用bionumberics软件分析DGGE结果,发现第一天和第二天样本的相似度最高为0.75,第三天与第一天相比,相似度为0.47,第四天与第三天相比,相似度又下降为0.16,表明禽肉在储存过程中不断有新的菌种出现,但是具体是什么菌种有待于高通量测序分析. 相似文献
6.
Frankia是共生、固氮并结瘤的放线菌,在其分类研究中,发现Frankia菌群中有丰富的遗传多样性.为了检测与考察这种多样性,我们采用16S-23S rDNA间隔区扩增产物的分析来研究弗兰克氏菌的遗传多样性. 相似文献
7.
生物脱硫技术在能源工业发展和环境保护等方面显示出潜在的优势。本文利用NaAc法抽取总DNA作模板，用真细菌专一性引物进行PCR方法扩增HB2的16S rDNA片段并进行琼脂糖凝胶的回收，选用PUCm—T—vector，用T4连接酶连接，转化大肠杆菌DH5α的感受态细胞，然后在含氨卞青霉素Ap／IPTG／X2gal的平板上筛选阳性克隆，PCR检测阳性克隆最后进行测序。结果表明，所测片段序列与多种假单胞杆菌的16S rDNA的同源性达98％以上。 相似文献
8.
阚劲松 《合肥联合大学学报》2003,13(4):93-98
应用PCR技术，从大肠杆菌中分别扩增出16S、23S核糖体RNA基因(rDNA)，以［a-^32P]bATP经缺口平移法标记rDNA后作为广谱探针。选用BamhI、EcoRI、HindⅢ等不同限制酶，对肠杆菌科细菌及一起沙门氏菌食物中毒暴发分离株进行染色体DNA酶切。凝胶电泳分离各片段后，通过Southern杂交。获得各菌株rDNA指纹图。每个细菌都可以从rDNA的限制性片段长度多态性(RFLP)得到鉴定。rDNA指纹图显示出种特异性。明确地区分出引起食物中毒的可疑传染源。该法特异性强、重复性好，既可用于细菌的分类鉴定。也可作为分子流行病学调查的工具。 相似文献
9.
DGGE和qPCR联用定量分析环境样品中优势菌群 总被引：1，自引：0，他引：1
精确定量环境样品中特定微生物类群的数量对阐明环境生物治理/修复过程中微生物的功能和效应具有重要意义.该文以土壤样品为例,将PCR-DGGE技术与定量PCR(qPCR)技术联用,定量检测样品中优势微生物类群数量.方法:抽提样品DNA,针对16S rDNA进行PCR-DGGE分析,明确样品中的优势菌群;再根据特定优势菌群的基因序列设计适合的定量引物,通过针对特定优势菌群的定量PCR分析,得到样品中优势菌群的数量信息.该方法灵敏度高、重复性好,并且无需培养,最大限度地保持了样品原始群落信息. 相似文献
10.
《昭通师范高等专科学校学报》2019,(5):39-45
通过现有恒河猴(Macaca mulatta,Mm)和藏酋猴(Macaca thibetana,Mt)肠道微生物的16SrRNA高通量测序鉴定结果进行对比分析;结果显示,恒河猴和藏酋猴肠道微生物门分类水平中的核心菌群包括Firmicutes、Bacteriodetes、Proteobacteria、Actinobacteria和Spirochaetia,占藏酋猴肠道微生物总量的95.81%;占恒河猴肠道微生物总量的90.87%。在属分类水平中核心菌群包括Catenibacterium、Dialister、Faecalibacterium、Blautia、Dorea、Roseburia和Helicobacter,占藏酋猴肠道微生物总量的27.43%;占恒河猴肠道微生物总量的9.31%。从门分类水平和属分类水平中,猴属动物肠道微生物共有核心菌群所占比例显著大于种群特有菌群,但在不同物种中丰富度存在显著差异;目前研究表明上述共有菌群大多与动物生活环境和食物资源存在一定联系;或许这也表明动物适应性进化过程中,环境和食性等其他因素对动物肠道微生物组成结构的影响更大。 相似文献
11.
The clinical diagnosis of sepsis is difficult, particularly in neonates. To devise a rapid and reliable method for identifing
bacteria in blood and cerebrospinal fluid (CSF), we developed a pair of primers according to the gene encoding 16 s rRNA,
found in all bacteria. DNA fragments from different bacterial species and from clinical samples were detected with polymerase
chain reaction (PCR), and with reverse hybridization using a universal bacterial probe, a gram-positive probe and a gram-negative
probe. Our results showed that a 371 bp DNA fragment was amplified from 20 different bacterial species. No signal was observed
when human DNA and viruses were used as templates. The sensitivity could be improved to 10−12 g. All 26 culture-positive clinical samples (22 blood samples and 4 CSF samples), were positive with PCR. The gram-negative
and gram-positive probe hybridized to clinical samples and to known bacterial controls, as predicted by Gram’s stain characteristics.
Our results suggest that the method of PCR and reverse hybridization is rapid, sensitive and specific in detecting bacterial
infections. This finding may be significant in the clinical diagnosis of sepsis in neonates.
Project (396457) supported by the Zhejiang Provincal Natural Science Foundation. 相似文献
12.
Jun Zhao Xin Zhao Lei Chao Wei Zhang Tao You Jie Zhang 《Journal of Zhejiang University. Science. B》2014,15(7):670-680
Pollution discharge disturbs the natural functions of water systems. The environmental microbial community composition and diversity are sensitive key indicators to the impact of water pollutant on the microbial ecology system over time. It is meaningful to develop a way to identify the microbial diversity related to heavy metal effects in evaluating river pollution. Water and sediment samples were collected from eight sections along the Tiaozi River where wastewater and sewage were discharged from Siping City in northeastern China. The main pollutants contents and microbial communities were analyzed. As the primary metal pollutants, zinc (Zn) and arsenic (As) were recorded at the maximum concentrations of 420 and 5.72 μg/L in the water, and 1704 and 1.92 mg/kg in the sediment, respectively. These pollutants posed a threat to the microbial community diversity as only a few species of bacteria and eukaryotes with strong resistance were detected through denaturing gradient gel electrophoresis (DGGE). Acinetobacter johnsonii, Clostridium cellulovorans, and Trichococcus pasteurii were the dominant bacteria in the severely polluted areas. The massive reproduction of Limnodrilus hoffmeisteri almost depleted the dissolved oxygen (DO) and resulted in the decline of the aerobic bacteria. It was noted that the pollution reduced the microbial diversity but the L. hoffmeisteri mass increased as the dominant community, which led to the overconsuming of DO and anaerobic stinking water bodies. Water quality, concentrations of heavy metals, and the spatial distribution of microbial populations have obvious consistencies, which mean that the heavy metals in the river pose a serious stress on the microorganisms. 相似文献
13.
本研究以NS5-NS6为引物,扩增真菌同源性序列ssu-rDNA片段,建立广范围真菌检测的方法。用此方法扩增医学主要致病真菌、细菌和人体细胞,结果所有真菌均扩增出一个约310bp的产物,而细菌和人体细胞均扩增阴性,1pg白色假丝酵母菌DNA即可检出。 相似文献
14.
Detection of PCV2 DNA by SYBR Green I-based quantitative PCR 总被引：5，自引：0，他引：5
We developed an assay for the detection and quantitation of porcine circovirus type 2 (PCV2) with the SYBR Green I-based real-time PCR. The real-time PCR provides a broad dynamic range, detecting from 103 to 1011 copies of DNA per reaction. No cross-reactions were found in specimens containing PCV1. Because of the high sensitivity and specificity of the assay with a relatively rapid and simple procedure, real-time PCR can be used as a routine assay for the clinical diagnosis of PCV2 infection. In this study we applied real-time PCR assay to 80 clinical samples, collected from 40 pigs with postweaning multisystemic wasting syndrome (PMWS) and 40 healthy pigs in comparison with conventional PCR assay. In 56 of 80 samples, PCV2 DNA was de-tected by conventional PCR assay. All samples positive for PCV2 DNA in conventional PCR assay were also positive in real-time assay, and 12 of 24 samples that tested negative for PCV2 DNA in the conventional assay were tested positive in real-time PCR assay. Real-time PCR assay increased the number of samples in which PCV2 was detected by 15%. It is, therefore, considered to be a useful tool for the detection of PCV2. 相似文献
15.
In the search for a rapid and reliable method for identification of bacteria in blood and cerebrospinal fluid , we developed a unified set of primers and used them under polymerase chain reaction(PCR) to amplify the spacer regions between the 16s and 23s genes in the prokaryotic rRNA genetic loci . Spacer regions within these loci showed a significant level of length and sequence polymorphism across most of the species lines. A generic pair of priming sequences was selected from highly conserved sequences in the 16s and 23s genes occurring adjacent to these polymorphic regions. This single set of primers and reaction conditions were used for the amplification of the 16s-23s spacer regions for 61 strains of standard bacteria and corresponding clinical isolates belonging to 20 genera and 27 species, including Listeria, Staphylococcus and Salmonella species, et al. When the spacer amplification products were resolved by electrophoresis, the resulting patterns could be used to distinguish most of the bacteria species within the test group, and the amplification products of the clinical isolates clustered at the standard species level. Some species presenting similar pattern were further analyzed by HinfI or AluI digestion or DNA clone and sequences analysis in order to establish the specific 16s-23s rRNA gene spacer regions map. Analysis of 42 blood specimens from septicemic neonates and 6 CSF specimens from suspected purulent meningitis patients by bacterial culture and PCR-RFLP(Restriction Fregament Length Polymorphism) showed that 15 specimens of blood culture were positive(35.7%) in the 42 septicemic neonates; 27 specimens were positive(64.2%) by PCR, and that the positive rate by PCR was significantly higher than that by blood culture(P<0.01). Among the 6 CSF specimens, one specimen found positive by blood culture was also positive by PCR, two found negative by blood culture showed positive by PCR; all three were S.epidermidis according to the DNA map. One C.neoformans found positive by blood culture showed negative by PCR. The remaining two specimens were both negative by PCR and blood culture. These results indicated that the method of detecting bacterial 16s-23s rRNA spacer regions using PCR and RFLP techniques was rapid, sensitive and specific in the detection of bacterial infections; and so, has very important application in the clinical diagnosis of sepsis in neonates. 相似文献
16.
Bao-zhong Zhang Xin Zhang Xiao-ping An Duo-liang Ran Yu-sen Zhou Jun Lu Yi-gang Tong 《Journal of Zhejiang University. Science. B》2009,10(6):479-482
Site-directed mutagenesis (SDM) has been a very important method to probe the function-structure relationship of proteins. In this study, we introduced an easy-to-use, polymerase chain reaction (PCR)-based SDM method for double-stranded plasmid DNA, with a designed restriction site to ensure simple and efficient mutant screening. The DNA sequence to be mutated was first translated into amino acid sequence and then the amino acid sequence was reversely translated into DNA sequence with degenerate codons, resulting in a large number of sequences with silent mutations, which contained various restriction endonuclease (RE) sites. Certain mutated sequence with an appropriate RE site was selected as the target DNA sequence for designing a pair of mutation primers to amplify the full-length plasmid via inverse PCR. The amplified product was 5'-phosphorylated, circularized, and transformed into an Escherichia coli host. The transformants were screened by digesting with the designed RE. This protocol uses only one pair of primers and only one PCR is conducted, without the need for hybridization with hazardous isotope for mutant screening or subcloning step. 相似文献
17.
Xu Lei Wu Zhifang Wang Yuan Wang Sa Shu Chang Duan Zhuhui Deng Shuli 《Journal of Zhejiang University. Science. B》2021,22(4):285-294
Objectives: The study aimed at identifying salivary microbiota in caries-free Chinese preschool children using highthroughput sequencing. Methods: Saliva samples were obtained from 35 caries-free preschool children(18 boys and 17 girls)with primary dentition, and 16 S ribosomal DNA(r DNA) V3–V4 hypervariable regions of the microorganisms were analyzed using Illumina MiSeq. Results: At 97% similarity level, all of these reads were clustered into 334 operational taxonomic units(OTUs). Among these, five phyla(Firmicutes, Proteobacteria, Actinobacteria, Bacteroidetes, and Candidate division TM7) and13 genera(Streptococcus, Rothia, Granulicatella, Prevotella, Enterobacter, Veillonella, Neisseria, Staphylococcus, Janthinobacterium, Pseudomonas, Brevundimonas, Devosia, and Gemella) were the most dominant, constituting 99.4% and 89.9% of the salivary microbiota, respectively. The core salivary microbiome comprised nine genera(Actinomyces, Capnocytophaga, Gemella,Granulicatella, Lachnoanaerobaculum, Neisseria, Porphyromonas, Rothia, and Streptococcus). Analysis of microbial diversity and community structure revealed a similar pattern between male and female subjects. The difference in microbial community composition between them was mainly attributed to Neisseria(P=0.023). Furthermore, functional prediction revealed that the most abundant genes were related to amino acid transport and metabolism. Conclusions: Our results revealed the diversity and composition of salivary microbiota in caries-free preschool children, with little difference between male and female subjects.Identity of the core microbiome, coupled with prediction of gene function, deepens our understanding of oral microbiota in cariesfree populations and provides basic information for associating salivary microecology and oral health. 相似文献