920 resultados para ISSR-PCR


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应用ISSR分子标记揭示了5种缘毛类纤毛虫(Carchesium polypinum,Epistylis chrysemydis,E.plicatilis,E.urceolata和Vorticella campanula)的遗传关系。从34个引物中筛选到13个多态性高的引物进行研究。得到的遗传距离(0.6667~1.0000)显示ISSR技术具有较高的分辨率。依据构建的UPGMA聚类树,V.campanula首先和其他种类分开;C.polypinum和E.chrysemydis聚在了一起;在3种Epist

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[目的]筛选和优化高原鼠兔(Ochotona curzoniae)适宜的ISSR反应体系,以在对高原鼠兔进行ISSR分析时获得清晰和多态性好的扩增结果。[方法]以高原鼠兔基因组DNA为模板,通过单因素试验,对体系中的模板浓度、Mg2+浓度、dNTPs浓度、Taq酶用量、引物用量、退火温度进行探讨。[结果]结果表明,高原鼠兔ISSR-PCR扩增的最佳条件为:25μl PCR反应体系,其中4lμDNA模板,1.5 mmol/LMgCl2,0.2 mmol/L dNTPs,1.25 UTaq聚合酶,1.5μmol/L引物,复性温度4560℃(退火温度随引物不同而确定)。用11条引物进行了PCR扩增,筛选出效果较好的6条引物。[结论]该反应体系的建立为鼠兔遗传多样性和分子系统学研究提供技术支持。

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杜鹃花属(Rhododendron L.)植物分布广泛,研究发现所有的杜鹃花科植物都能形成一种特殊的菌根——杜鹃花类菌根(Ericoid Mycorrhiza)。杜鹃花类菌根对杜鹃花科植物在营养胁迫的环境下生长起到重要的作用。近几年,对杜鹃花类菌根的生物学和生态功能的研究越来越重视。我国是杜鹃花属植物资源最为丰富的国家,因此研究杜鹃花属植物菌根真菌多样性,充分利用杜鹃花特有的菌根资源,促进杜鹃花迁地保护成功具有重大的意义。 本研究以分布较广并且是中国特有的杜鹃花属植物——大白花杜鹃(Rhododendron decorum Franch.)的野生植株为研究对象,应用直接扩增根中真菌ITS区的分子鉴定方法和T-RFLP(末端限制性片段长度多态性)的分析方法,来研究其菌根真菌的多样性;并结合生态化学计量学特征分析、宿主遗传相似性及其群落组成分析等内容,探讨大白花杜鹃的菌根真菌-宿主植物-根际土壤三者之间的关系。主要结果如下: (1)通过用直接扩增真菌ITS区序列,揭示了大白花杜鹃根部真菌的多样性,本研究发现,野生大白杜鹃根部的真菌种类比较丰富,至少有26个ITS-taxa,包括子囊菌和担子菌共5个真菌目:Helotiales、Lecanorales(≡Agyriales)、Onygenales、Sebacinales和Thelephorales,其中包括典型的ERM真菌——树粉孢属Oidiodendron sp.(Myxotrichaceae)真菌。另外还发现了黑色有隔内生菌(Dark septate endophyte,DSE)以及一些未命名的子囊菌。担子菌在本研究中占有较大比例,尤其是蜡壳耳菌目真菌;此外还有较典型的外生菌根真菌——革菌目真菌。 (2)大白花杜鹃野生植株与栽培植株在菌根真菌种类组成上,有一定的相似性;在忽略种源差异等条件下相较而言,前者的物种丰富度远高于后者。 (3)大白花杜鹃菌根真菌多样性和丰富度同它的根际土壤与叶片的C、N、P含量以及C/N、N/P、土壤pH值、宿主的海拔高度等都没有显著的相关关系。 (4)在大白花杜鹃的菌根真菌群落组成方面,整体上保持了相当程度的相似性,同时还保持了一定水平的差异;大白杜鹃菌根真菌的种类是丰富的,优势度指数表明其多样性水平很高。 (5)大白花杜鹃的遗传距离与其菌根真菌群落组成结构有极显著的相关关系,宿主的种内遗传差异可能对菌根真菌群落物种组成产生选择偏好。 (6)大白花杜鹃的群落组成与其菌根真菌群落组成有极为显著的关联性,伴生种的菌根类型可能会影响宿主植物菌根真菌的物种组成结构。

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ISSR是一种特异性较强、稳定性较高的分子标记方法。本研究采用ISSR-PCR技术,检测了红松(PinuskoraiensisSieb.etZucc)在伊春市汤旺河高峰林场、长白山二道白河、黑河胜山林场和俄罗斯海参崴市郊的四个种群的遗传多样性和遗传分化。15个引物的扩增结果表明:红松群体的多态位点比率是60.70%,平均每个引物3.6个多态位点,多样性水平在松科植物中是较高的;红松分布中心区的遗传多样性要高于边界区;和大多数木本植物一致,红松群体的基因多样性主要来自种群内部,占总基因多样性的73%;红松四个种群的遗传距离和地理距离之间无正相关性。根据研究结果推测,天然红松分布区逐渐缩小的原因不是由于遗传多样性水平过低引起的,而是由于人类的破坏作用,再加上火灾和风倒等因素造成的。

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Pós-graduação em Agronomia (Entomologia Agrícola) - FCAV

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Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

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采用ISSR分子标记对中国水域隶属于不同江豚(Neophocaena phocaenoides)亚种的两个群体即长江群体和渤海群体的遗传多样性进行分析。用19条ISSR引物及3对引物组合对两个群体共36个样品的基因组DNA进行PCR扩增,共得到115个清晰的扩增位点,其中多态性位点48个,多态位点百分率(PPL)为41.71%。POPGENE分析结果表明:两个江豚群体的总体遗传多样性水平相对较低(He=0.1643;Ho=0.2413);长江群体的遗传多样性水平(He=0.1530;Ho=0.2223)稍

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Fragaria vesca is a short-lived perennial with a seasonal-flowering habit. Seasonality of flowering is widespread in the Rosaceae and is also found in the majority of temperate polycarpic perennials. Genetic analysis has shown that seasonal flowering is controlled by a single gene in F. vesca, the SEASONAL FLOWERING LOCUS (SFL). Here, we report progress towards the marker-assisted selection and positional cloning of SFL, in which three ISSR markers linked to SFL were converted to locus-specific sequence-characterized amplified region (SCAR1–SCAR3) markers to allow large-scale screening of mapping progenies. We believe this is the first study describing the development of SCAR markers from ISSR profiles. The work also provides useful insight into the nature of polymorphisms generated by the ISSR marker system. Our results indicate that the ISSR polymorphisms originally detected were probably caused by point mutations in the positions targeted by primer anchors (causing differential PCR failure), by indels within the amplicon (leading to variation in amplicon size) and by internal sequence differences (leading to variation in DNA folding and so in band mobility). The cause of the original ISSR polymorphism was important in the selection of appropriate strategies for SCAR-marker development. The SCAR markers produced were mapped using a F. vesca f. vesca × F. vesca f. semperflorens testcross population. Marker SCAR2 was inseparable from the SFL, whereas SCAR1 mapped 3.0 cM to the north of the gene and SCAR3 1.7 cM to its south.

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The prevalence and concentrations of Campylobacter jejuni, Salmonella spp. and enterohaemorrhagic E. coli (EHEC) were investigated in surface waters in Brisbane, Australia using quantitative PCR (qPCR) based methodologies. Water samples were collected from Brisbane City Botanic Gardens (CBG) Pond, and two urban tidal creeks (i.e., Oxley Creek and Blunder Creek). Of the 32 water samples collected, 8 (25%), 1 (3%), 9 (28%), 14 (44%), and 15 (47%) were positive for C. jejuni mapA, Salmonella invA, EHEC O157 LPS, EHEC VT1, and EHEC VT2 genes, respectively. The presence/absence of the potential pathogens did not correlate with either E. coli or enterococci concentrations as determined by binary logistic regression. In conclusion, the high prevalence, and concentrations of potential zoonotic pathogens along with the concentrations of one or more fecal indicators in surface water samples indicate a poor level of microbial quality of surface water, and could represent a significant health risk to users. The results from the current study would provide valuable information to the water quality managers in terms of minimizing the risk from pathogens in surface waters.

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The host specificity of the five published sewage-associated Bacteroides markers (i.e., HF183, BacHum, HuBac, BacH and Human-Bac) was evaluated in Southeast Queensland, Australia by testing fecal DNA samples (n = 186) from 11 animal species including human fecal samples collected via influent to a sewage treatment plant (STP). All human fecal samples (n = 50) were positive for all five markers indicating 100% sensitivity of these markers. The overall specificity of the HF183 markers to differentiate between humans and animals was 99%. The specificities of the BacHum and BacH markers were > 94%, suggesting that these markers are suitable for sewage pollution in environmental waters in Australia. The BacHum (i.e., 63% specificity) and Human-Bac (i.e., 79% specificity) markers performed poorly in distinguishing between the sources of human and animal fecal samples. It is recommended that the specificity of the sewage-associated markers must be rigorously tested prior to its application to identify the sources of fecal pollution in environmental waters.

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Aims: Influenza is commonly spread by infectious aerosols; however, detection of viruses in aerosols is not sensitive enough to confirm the characteristics of virus aerosols. The aim of this study was to develop an assay for respiratory viruses sufficiently sensitive to be used in epidemiological studies. Method: A two-step, nested real-time PCR assay was developed for MS2 bacteriophage, and for influenza A and B, parainfluenza 1 and human respiratory syncytial virus. Outer primer pairs were designed to nest each existing real-time PCR assay. The sensitivities of the nested real-time PCR assays were compared to those of existing real-time PCR assays. Both assays were applied in an aerosol study to compare their detection limits in air samples. Conclusions: The nested real-time PCR assays were found to be several logs more sensitive than the real-time PCR assays, with lower levels of virus detected at lower Ct values. The nested real-time PCR assay successfully detected MS2 in air samples, whereas the real-time assay did not. Significance and Impact of the Study: The sensitive assays for respiratory viruses will permit further research using air samples from naturally generated virus aerosols. This will inform current knowledge regarding the risks associated with the spread of viruses through aerosol transmission.

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Staphylococcus aureus is a common pathogen that causes a variety of infections including soft tissue infections, impetigo, septicemia toxic shock and scalded skin syndrome. Traditionally, Methicillin-Resistant Staphylococcus aureus (MRSA) was considered a Hospital-Acquired (HA) infection. It is now recognised that the frequency of infections with MRSA is increasing in the community, and that these infections are not originating from hospital environments. A 2007 report by the Centers for Disease Control and Prevention (CDC) stated that Staphylococcus aureus is the most important cause of serious and fatal infections in the USA. Community-Acquired MRSA (CA-MRSA) are genetically diverse and distinct, meaning they are able to be identified and tracked by way of genotyping. Genotyping of MRSA using Single nucleotide polymorphisms (SNPs) is a rapid and robust method for monitoring MRSA, specifically ST93 (Queensland Clone) dissemination in the community. It has been shown that a large proportion of CA-MRSA infections in Queensland and New South Wales are caused by ST93. The rationale for this project was that SNP analysis of MLST genes is a rapid and cost-effective method for genotyping and monitoring MRSA dissemination in the community. In this study, 16 different sequence types (ST) were identified with 41% of isolates identified as ST93 making it the predominate clone. Males and Females were infected equally with an average patient age of 45yrs. Phenotypically, all of the ST93 had an identical antimicrobial resistance pattern. They were resistant to the β-lactams – Penicillin, Flu(di)cloxacillin and Cephalothin but sensitive to all other antibiotics tested. Virulence factors play an important role in allowing S. aureus to cause disease by way of colonising, replication and damage to the host. One virulence factor of particular interest is the toxin Panton-Valentine leukocidin (PVL), which is composed of two separate proteins encoded by two adjacent genes. PVL positive CA-MRSA are shown to cause recurrent, chronic or severe skin and soft tissue infections. As a result, it is important that PVL positive CA-MRSA is genotyped and tracked. Especially now that CA-MRSA infections are more prevalent than HA-MRSA infections and are now deemed endemic in Australia. 98% of all isolates in this study tested positive for the PVL toxin gene. This study showed that PVL is present in many different community based ST, not just ST93, which were all PVL positive. With this toxin becoming entrenched in CA-MRSA, genotyping would provide more accurate data and a way of tracking the dissemination. PVL gene can be sub-typed using an allele-specific Real-Time PCR (RT-PCR) followed by High resolution meltanalysis. This allows the identification of PVL subtypes within the CA-MRSA population and allow the tracking of these clones in the community.

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This paper aimed to assess the magnitude of sewage pollution in an urban lake in Dhaka, Bangladesh by using Quantitative PCR (qPCR) of sewage-associated Bacteroides HF183 markers. PCR was also used for the quantitative detection of ruminant wastewater-associated CF128 markers along with the enumeration of traditional fecal indicator bacteria, namely, enterococci. The number of enterococci in lake water samples ranged from 1.1 x 104 to 1.9 x 105 CFU/100 ml of water. From the 20 water samples tested, 14 (70%) and 7 (35%) were PCR positive for the HF183 and CF128 markers, respectively. The numbers of the HF183 and CF128 markers in lake water samples were 3.9 x 104 to 6.3 × 107 and 9.3 x 103 to 6.3 x 105 genomic units (GU)/100 ml of water, respectively. The high numbers of enterococci and the HF183 markers indicate sewage pollution and potential health risks to those who use the lake water for non-potable purposes such as bathing and washing clothes. This is the first study that investigated the presence of microbial source tracking (MST) markers in Dhaka, Bangladesh where diarrhoeal diseases is one of the major causes of childhood mortality. The molecular assay as used in this study can provide valuable information on the extent of sewage pollution, thus facilitating the development of robust strategies to minimise potential health risks.

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In total, 782 Escherichia coli strains originating from various host sources have been analyzed in this study by using a highly discriminatory single-nucleotide polymorphism (SNP) approach. A set of eight SNPs, with a discrimination value (Simpson's index of diversity [D]) of 0.96, was determined using the Minimum SNPs software, based on sequences of housekeeping genes from the E. coli multilocus sequence typing (MLST) database. Allele-specific real-time PCR was used to screen 114 E. coli isolates from various fecal sources in Southeast Queensland (SEQ). The combined analysis of both the MLST database and SEQ E. coli isolates using eight high-D SNPs resolved the isolates into 74 SNP profiles. The data obtained suggest that SNP typing is a promising approach for the discrimination of host-specific groups and allows for the identification of human-specific E. coli in environmental samples. However, a more diverse E. coli collection is required to determine animal- and environment-specific E. coli SNP profiles due to the abundance of human E. coli strains (56%) in the MLST database.