反向PCR方法克隆腾冲嗜酸两面菌的分子伴侣基因*

根据已知的Sulfolobus属的分子伴侣基因,设计简并引物,用PCR的方法从腾冲嗜酸两面菌(Acidianus tengchongensis)基因组DNA中分别克隆到了分子伴侣α亚基和β亚基的约500bp的基因片段。以它们为探针进行Southern杂交,确定了合适的限制性内切酶。以确定的限制性内切酶消化的基因组DNA环化物为模板,进行反向PCR反应,引物的延伸方向由已知序列出发沿环化分子向未知区域进行,扩增产物经测序表明为α亚基和β亚基基因。根据所得序列分别设计两对引物进行PCR,测序结果     

利用长引物嵌套式反向PCR方法克隆雅致枝霉Δ6-脂肪酸脱氢酶基因上游序列

用限制酶EcoRⅠ、KpnⅠ分别对雅致枝霉As32806基因组DNA进行消化,而后在低浓度条件下利用T4DNA连接酶使DNA自身环化。根据已知基因序列,设计一对长度为35nt的长反向引物和两对较短的引物,以基因组连接产物为模板,通过三轮嵌套式PCR反应,获得一长度约为4kb的扩增片段。经序列测定表明得到了Δ⁶-脂肪酸脱氢酶基因上游序列约为1.3kb,初步序列分析显示该序列为一潜在的启动子序列。     

利用长引物嵌套式反向PCR方法克隆雅致枝霉Δ6-脂肪酸脱氢酶基因上游序列

用限制酶EcoRⅠ、KpnⅠ分别对雅致枝霉As3.2806基因组DNA进行消化,而后在低浓度条件下利用T4DNA连接酶使DNA自身环化。根据已知基因序列,设计一对长度为35nt的长反向引物和两对较短的引物,以基因组连接产物为模板,通过三轮嵌套式PCR反应,获得一长度约为4kb的扩增片段。经序列测定表明得到了Δ6-脂肪酸脱氢酶基因上游序列约为1.3kb,初步序列分析显示该序列为一潜在的启动子序列。     

环状芽孢杆菌C-2几丁酶基因的克隆

环状芽孢杆菌(Bacillus circulans)C-2总DNA经PstI部分酶切后分离2～10kb的片段,插入质粒pUC19的PstI位点,转化大肠杆菌(Escherichia coli),利用几丁质平板从约8000个重组子中筛选到一个几丁酶基因阳性克隆(命名为pCHT1)。用12种限制酶对重组质粒进行的酶切分析表明,重组质粒中的插入片段长3.0kb,其中各有一个KpnI,SacI和SspI位点。把该克隆片段反向插入pUC19的PstI位点所得到的重组子同样具有几丁酶基因表达活性,说明此片段含有一个完整的几丁酶基因,其自身的启动子能被大肠杆菌转录系统所识别。Southern杂交证实了该片段来自于B.circulans C-2基因组,且以单拷贝形式存在,它不能与来自于其它7株几丁酶产生菌的总DNA杂交。     

几种固氮菌nifA基因片段的同源性分析

参照已知数种固氮菌nifA基因的DNA序列,选择其中间区域的两个保守性较强的序列合成引物,利用肺炎克匠杆菌(Klebsiella pneumoniaef)、棕色固氮菌(Azotobacter vinela-ndii)、巴西固氮螺菌(Azospirllum brasilense)、草螺菌(Herbaspirillum seropedicae)和深红红螺菌(Rhodospirillum rubrum)的总DNA进行聚合酶链反应,结果均扩增出约450bp大小的片段,经证实为各种固氮菌的nifA基因部分片段。 核酸印迹分子杂交结果显示出nifA基因在不同固氮菌中的同源性不强。     

大肠杆菌Na+/H+反向运输体A基因的克隆及序列分析*

为研究细菌的耐盐机理 ,根据细菌中存在的Na⁺/H⁺反向运输体 (nhaA)的基因序列 ,采用PCR扩增的方法 ,从大肠杆菌 (Escherichiacoli)DH5α中克隆获得了11kb的DNA片段 ,经过核酸序列分析 ,发现基因片段包含了nhaA基因完整的读码框架。采用序列同源性分析方法 ,结果显示nhaA基因在多种细菌中均存在 ,表明nhaA基因是细菌中普遍存在的一种能够反向运输Na^{+相似文献}   

采用苯酚羟化酶基因特异引物检测苯酚降解菌

根据苯酚羟化酶基因高度保守序列设计了一对该基因的特异PCR引物。采用该特异引物从苯酚降解菌醋酸钙不动杆菌 (Acinetobactercalcoaceticus)PHEA 2的总DNA中扩增到唯一一条大小为 684bp的片段。该DNA片段与已知的A .calcoaceticusNCIB82 50的苯酚羟化酶基因具有高度的同源性 ,其核苷酸序列的同源性为 84% ,推导的氨基酸序列的同源性为 98%。对苯酚和非苯酚降解菌株的PCR扩增结果表明 :所有苯酚降解菌均能扩增出 684bp的特征片段 ,而非苯酚降解菌则无PCR条带。对炼焦废水中的细菌群落进行PCR扩增和生化特性检测表明 :显示 684bp特征片段的菌株均具有苯酚降解特性。上述结果表明 ,利用苯酚羟化酶基因的特异引物可对环境中的苯酚降解菌株进行准确快速的PCR检测。     

反向PCR方法克隆腾冲嗜酸两面菌的分子伴侣基因

根据已知的Sulfolobus属的分子伴侣基因,设计简并引物,用PCR的方法从腾冲嗜酸两面菌（Acidianus tengchongensis）基因组DNA中分别克隆到了分子伴侣α亚基和β亚基的约500bp的基因片段。以它们为探针进行Southern杂交,确定了合适的限制性内切酶。以确定的限制性内切酶消化的基因组DNA环化物为模板,进行反向PCR反应,引物的延伸方向由已知序列出发沿环化分子向未知区域进行,扩增产物经测序表明为α亚基和β亚基基因。根据所得序列分别设计两对引物进行PCR,测序结果表明得到了α亚基和β亚基的完整基因。     

串珠镰孢霉D-泛解酸内酯水解酶基因的克隆及在大肠杆菌中的表达

利用D_泛解酸内酯水解酶N末端序列,并根据NCBI中公布的D_泛解酸内酯水解酶cDNA序列设计了一个特异引物,该引物结合Oligo(dT) 1 5,以串珠镰孢霉(Fusariummoniliforme)CGMCC 0 5 36mRNA反转录得到的总cDNA为模板进行扩增,获得约1 5kb左右的片段,将其克隆到T载体上进行测序,对测得的序列进行分析,重新设计了一对引物,并在引物两端分别加上限制酶EcoRⅠ和SalⅠ的识别位点序列,利用热启动PCR成功地扩增出了D_泛解酸内酯水解酶基因,基因片段长度为114 6bp ,该序列同来源于尖镰孢霉菌(F .oxysporum)AKU 370 2菌株的编码D_泛解酸内酯水解酶cDNA结构基因的同源性为90 0 6 %。将所得片段定向克隆到pTrc99a载体中,转化至JM10 9感受态细胞,筛选出了阳性克隆。经IPTG诱导阳性菌,进行SDS_PAGE电泳,检测出在约4 0kD处有一蛋白表达带。对两株重组基因工程菌的比活力进行测定,结果分别为37U和4 1U。     

Detection of the Bartonella DNA by the method of nested PCR in patients after tick bites in Novosibirsk region

Arthropod-borne bacterial pathogen Bartonella DNA was detected in human blood cells after tick bites in summer 2003 and 2004 in Novosibirsk region by nested PCR with primers specific to groEL gene of HSP60 protein. Comparative assay of 190 p.b. of long PCR fragment revealed that the nucleotide sequences might belong to Bartonella henselae and Bartonella quintana.     

Molecular differentiation of Bifidobacterium species with amplified ribosomal DNA restriction analysis and alignment of short regions of the ldh gene

The differentiation of Bifidobacterium species was performed with specific primers using the PCR technique, the amplified ribosomal DNA restriction analysis (ARDRA) technique based on reports on the sequence of the 16S rRNA gene and speciation based on a short region of the ldh gene. Four specific primer sets were developed for each of the Bifidobacterium species, B. animalis, B. infantis and B. longum. The use of the ARDRA method made it possible to discriminate between B. infantis, B. longum and B. animalis with the combination of BamHI, TaqI and Sau3AI restriction enzymes. The ldh gene sequences of 309-312 bp were determined for 19 Bifidobacterium strains. Alignment of these short regions of the ldh gene confirmed that it is possible to distinguish between B. longum and B. infantis but not between B. lactis and B. animalis.     

Cloning of HSP60 (GroEL) operon from Clostridium perfringens using a polymerase chain reaction based approach.

Using degenerate oligonucleotide primers for conserved regions of HSP60, a 0.6 kilobase fragment of Clostridium perfringens DNA was amplified by the polymerase chain reaction. The amplified fragment was used as a probe to isolate a genomic clone containing the C. perfringens HSP60 operon. The clone contained two open reading frames homologous to the GroES and GroEL (or HSP60) family of bacterial and eukaryotic proteins as well as other upstream and downstream sequences. The approach described here, employing this set of degenerate oligonucleotide primers, could be used to clone HSP60 gene/cDNA from any species.     

双歧杆菌属特异性测序引物筛选及优化

【背景】目前双歧杆菌的益生功能被普遍认可,越来越多的研究开始关注肠道中双歧杆菌的生物多样性。然而双歧杆菌是肠道中的低丰度物种,现有技术尚难以深入研究其多样性。【目的】基于双歧杆菌16SrRNA基因序列筛选一对适用于分析粪便样品中低丰度双歧杆菌属多样性的特异性引物。【方法】依据已有引物的相对位置及其与双歧杆菌属16S rRNA基因序列的匹配率,将引物重组优化得到扩增片段800 bp的双歧杆菌属特异性引物;通过PCR扩增和琼脂糖凝胶电泳对引物进行实验筛选和特异性验证;以细菌通用引物(27f/1492r)为参照,通过单分子实时(Single-molecule real-time,SMRT)测序技术对不同引物的3份粪便样品中细菌的DNA扩增子进行测序,在种水平上分析比较不同引物的优劣。【结果】对文献中已有的9对双歧杆菌特异性引物进行重组并优化,其中2对引物的理论特异性较好且扩增产物大于800 bp,它们分别为Bif164-f/Pbi R2和Pbi F1/Pbi R2。PCR扩增和琼脂糖凝胶电泳实验发现,Bif164-f/Pbi R2的扩增条带明亮且无拖尾。此外,利用SMRT测序平台对引物27f/1492r和Bif164-f/Pbi R2的3份粪便样品中细菌的DNA扩增子进行测序并分析。27f/1492r扩增子的分析结果显示,3份样品依次分别含1、3、4个双歧杆菌种且双歧杆菌的平均相对含量为0.34%;而Bif164-f/Pbi R2扩增子的分析结果显示,3份样品依次分别含2、6和8个双歧杆菌种且双歧杆菌的平均相对含量为98.72%。上述结果表明,Bif164-f/Pbi R2可在种水平上特异地检出粪便中低丰度的双歧杆菌,进而实现样品中双歧杆菌的多样性分析。【结论】筛选出一对双歧杆菌特异性引物Bif164-f/PbiR2,可在种水平上分析粪便样品中低丰度双歧杆菌的生物多样性,同时也验证了理论结合实验进行引物筛选这种方法的可行性。     

Characterization of human intestinal bifidobacteria using competitive PCR and PCR-TTGE

In this study, a competitive PCR was developed to estimate the quantity of bifidobacteria in human faecal samples using two 16S rRNA gene Bifidobacterium genus-specific primers, Bif164f and Bif662r. A PCR-temporal temperature gradient gel electrophoresis (TTGE) with the same primers also allowed us to describe the Bifidobacterium species present in these faecal samples. The PCR product obtained from the competitor had 467 bp, and was 47 bp shorter than the PCR products obtained from Bifidobacterium strains. The number of bifidobacterial cells was linear from 10 to 10(8) cells per PCR assay. Taking into account the dilutions of the extracted DNA, the linear range was over 8 x 10(5) bifidobacteria g(-1) of faeces. Reproducibility was assessed from 10 independent DNA extractions from the same stool and the coefficient of variation was 0.5%. When the competitive PCR was compared with the culture method, a similar count of seven out of nine Bifidobacterium pure cultures were obtained, or had a difference inferior or equal to 1 log(10). In faecal samples, the enumeration of Bifidobacterium genus in most cases gave higher results with competitive PCR than with culture on selective Columbia-Beerens agar pH 5 (P < 0.05). In conclusion, this competitive PCR allows a rapid, highly specific and reproducible quantification of Bifidobacterium genus in faecal samples. TTGE fragments co-migrating with B. longum CIP64.63 fragment were found in 10 out of 11 faecal samples. Bifidobacterium adolescentis and B. bifidum were detected in five out of 11 subjects. Thus, cPCR and PCR-TTGE can be associated in order to characterize human faecal bifidobacteria.     

Analysis of a gene (vch) encoding hemolysin isolated and sequenced from Vibrio campbellii

This study describes the amplification, localization, and sequence analysis of a hemolysin gene from type strain V. campbellii NBRC 15631--the first report of a full-length hemolysin gene for the species. An amplicon ( approximately 600 bp) of polymerase chain reaction performed using V. campbellii DNA template and primers previously designed to target a fragment of V. harveyi hemolysin gene (vhh) was shotgun-cloned and sequenced, generating 576 bp nucleotide sequences of the V. campbellii hemolysin gene. PCR primers designed based on these initial sequences were used to amplify a 551-bp V. campbellii hemolysin gene fragment that was used as probe in Southern hybridization, which localized the complete hemolysin gene within a 3.5-kb HindIII restriction fragment of the V. campbellii genomic DNA. To obtain the remaining DNA sequences upstream and downstream of the 576-bp hemolysin gene sequences, inverse PCR was performed using a self-ligated (circularized) V. campbellii HindIII restriction fragment as the template and PCR primers designed to amplify flanking regions of the 576-bp gene fragment. Nucleotide sequences from the terminal regions of the 3.1-kb product of inverse PCR provided the flanking sequences, resulting in the complete sequence for the V. campbellii hemolysin gene. A VCH PCR primer set was designed to amplify a 1.3-kb region containing the entire hemolysin gene even from other V. campbellii strains, which was sequenced to confirm the V. campbellii hemolysin gene sequence. An open reading frame (ORF) of 1,254 bp (designated as vch) was identified, sharing 79% sequence identity with V. harveyi hemolysin gene vhh, representing 262 base substitutions between V. campbellii and V. harveyi. The deduced amino acid sequence of V. campbellii hemolysin (VCH) shows homologies to the V. harveyi hemolysin (VHH), thermolabile hemolysin of V. parahaemolyticus, proteins such as phospholipase of V. vulnificus and lecithinases of V. mimicus and V. cholerae. The VCH primer set did not produce any amplicon in PCR using V. harveyi DNA, and may therefore be used to distinguish environmental strains of V. campbellii from V. harveyi.     

Genomic species typing of acinetobacters by polymerase chain reaction amplification of the recA gene

Abstract The recA gene has been used as a target in screening for the presence of acinetobacters on the genospecies level and differentiation of relevant acinetobacter species from one another by PCR. Primers deduced from known recA gene sequences of Acinetobacter calcoaceticus and Neisseria gonorrhoeae allowed the amplification of DNAs from all Acinetobacter genospecies. The size of the amplified DNA fragment from all genospecies tested was approximately 435–500 bp relative to DNA size markers. The amplified products were examined further by restriction fragment length polymorphism (RFLP) analysis. Restriction analysis with only two enzymes, Mbo I and Hin fI, enabled us to identify all known genospecies. Since this method uses conserved recA gene sequences for primers, it is expected to be applicable for the identification of most bacterial species.     

Oligonucleotide primers for detection and amplification of the emoA gene encoding bacterial ethylenediaminetetraacetate monooxygenase

A system of primers was designed on the basis of analysis of nucleotide sequences of the emoA gene encoding ethylenediaminetetraacetate (EDTA) monooxygenase, which are deposited in GenBank. This system of primers makes it possible to obtain emoA gene fragments approximately 750 bp long for bacterial destructors of EDTA. Polymerase chain reaction (PCR) of total DNA isolated from enrichment and pure cultures showed that this system can be effectively used for detecting the emoA gene in representatives of Alpha- and Gammaproteobacteria. Partial sequences of emoA genes of bacteria of the genera Chelativorans and Stenotrophomonas, which are able to degrade this pollutant, have been sequenced and deposited in GenBank.     

High-throughput sequencing of 16S rRNA gene amplicons: effects of extraction procedure, primer length and annealing temperature

The analysis of 16S-rDNA sequences to assess the bacterial community composition of a sample is a widely used technique that has increased with the advent of high throughput sequencing. Although considerable effort has been devoted to identifying the most informative region of the 16S gene and the optimal informatics procedures to process the data, little attention has been paid to the PCR step, in particular annealing temperature and primer length. To address this, amplicons derived from 16S-rDNA were generated from chicken caecal content DNA using different annealing temperatures, primers and different DNA extraction procedures. The amplicons were pyrosequenced to determine the optimal protocols for capture of maximum bacterial diversity from a chicken caecal sample. Even at very low annealing temperatures there was little effect on the community structure, although the abundance of some OTUs such as Bifidobacterium increased. Using shorter primers did not reveal any novel OTUs but did change the community profile obtained. Mechanical disruption of the sample by bead beating had a significant effect on the results obtained, as did repeated freezing and thawing. In conclusion, existing primers and standard annealing temperatures captured as much diversity as lower annealing temperatures and shorter primers.     

Bacterial diversity in worker adults of Apis mellifera capensis and Apis mellifera scutellata (Insecta: Hymenoptera) assessed using 16S rRNA sequences

High-fidelity PCR of 16S rRNA sequences was used to identify bacteria associated with worker adults of the honeybee subspecies Apis mellifera capensis and Apis mellifera scutellata. An expected approximately 1.5-kb DNA band, representing almost the entire length of the 16S rRNA gene, was amplified from both subspecies and cloned. Ten unique sequences were obtained: one sequence each clustered with Bifidobacterium (Gram-positive eubacteria), Lactobacillus (Gram-positive eubacteria), and Gluconacetobacter (Gram-negative alpha-proteobacteria); two sequences each clustered with Simonsiella (beta-proteobacteria) and Serratia (gamma-proteobacteria); and three sequences each clustered with Bartonella (alpha-proteobacteria). Although the sequences relating to these six bacterial genera initially were obtained from either A. m. capensis or A. m. scutellata or both, newly designed honeybee-specific 16S rRNA primers subsequently amplified all sequences from all individual workers of both subspecies. Attempts to amplify these sequences from eggs have failed. However, the wsp primers designed to amplify Wolbachia DNA from arthropods, including these bees, consistently produced a 0.6-kb DNA band from individual eggs, indicating that amplifiable bacterial DNA was present. Hence, the 10 bacteria could have been acquired orally from workers or from other substrates. This screening of 16S rRNA sequences from A. m. capensis and A. m. scutellata found sequences related to Lactobacillus and Bifidobacterium which previously had been identified from other honeybee subspecies, as well as sequences related to Bartonella, Gluconacetobacter, Simonsiella/Neisseria, and Serratia, which have not been identified previously from honeybees.