PCR-based assay for detection of four coral pathogens

Several microorganisms have been identified as pathogenic agents responsible for various outbreaks of coral disease. Little has been learned about the exclusivity of a pathogen to given disease signs. Most pathogens have only been implicated within a subset of corals, leaving gaps in our knowledge of the host range and geographic extent of a given pathogen. PCR-based assays provide a rapid and inexpensive route for detection of pathogens. Pathogen-specific 16S rDNA primer sets were designed to target four identified coral pathogens: Aurantimonas coralicida, Serratia marcescens, Vibrio shilonii, and Vibrio coralliilyticus. Assays detected the presence of targets at concentrations of less than one cell per microliter. The assay was applied to 142 coral samples from the Florida Keys, Puerto Rico, and U.S. Virgin Islands as an in situ specificity test. Assays displayed a high-level of specificity, seemingly limited only by the resolution of the 16S rDNA.

Extraction, PCR amplification, and sequencing of mitochondrial DNA from scent mark and feces in the giant panda

To expand the feasibility of applying simple, efficient, non-invasive DNA preparation methods using samples that can be obtained from giant pandas living in the wild, we investigated the use of scent markings and fecal samples. Giant panda-specific oligonucleotide primers were used to amplify a portion of the mitochondrial DNA control region as well as a portion of the mitochondrial DNA cytochrome b gene and tRNA(Thr) gene region. A 196 base pair (bp) fragment in the control region and a 449 bp fragment in the cytochrome b gene and tRNA(Thr) gene were successfully amplified. Sequencing of polymerase chain reaction (PCR) products demonstrated that the two fragments are giant panda sequences. Furthermore, under simulated field conditions we found that DNA can be extracted from fecal samples aged as long as 3 months. Our results suggest that the scent mark and fecal samples are simple, efficient, and easily prepared DNA sources. (C) 1998 Wiley-Liss, Inc.

Characterization of Sarcocystis species in domestic animals using a PCR-RFLP analysis of variation in the 18S rRNA gene: a cost-effective and simple technique for routine species identification

Thirteen restriction endonucleases were used to investigate nucleotide sequence variation in the 18S rRNA DNA of 88 individuals from ten Sarcocystis taxa collected as cysts from their intermediate hosts, swine, cattle and water buffalo. A DNA sequence of

A PCR-based RFLP analysis of Sarcocystis cruzi (Protozoa : Sarcocystidae) in Yunnan province, PR china, reveals the water buffalo (Bubalus bubalis) as a natural intermediate host

A polymerase chain reaction-based restriction fragment length polymorphism (RFLP) approach is used to examine Sarcocystis cruzi-like taxa from the atypical intermediate host, water buffalo, in Yunnan, People's Republic of China. The loci examined lie with

麂属动物陈旧皮张标本的DNA提取及PCR扩增

用改进的方法从保存于本单位标本中提取DNA, 所得DNA片段的分子量从100bp到1kb以上。利用线粒体DNA细胞色素b通用引物和PCR技术。从小麂、印度麂、贡山麂、费氏麂、黑麂DNA中扩增出307bp的 细胞色素b特异片段(加上两端引物后长度为364bp)。用28种限制性内切酶对 新鲜血样和从陈旧皮张标本中所得扩增片段进行酶切分析, 发现只有4个酶(DraⅠ、xbaⅠ、HaeⅢ、HpaⅡ)在这个片段上有切点, 其中HaeⅢ和HapⅡ的识别位点在各种麂中有所不同。 图3参10

Investigation of polymorphism by PCR-RFLP method in Cobia fish Rachycentron canadum in the Persian Gulf and Oman Sea

Cobia is a native fish species in Iranian waters in the Persian Gulf and Sea of Oman and has a good internal and foreign market. This fish is a fast growing species and for this reason Iranian Fisheries is considering to go for it culture practices. To go for any utilization such as fishing from wild stocks or culture activities, needs a better understanding of its peculiarities and genetic characteristics of its natural resources. Therefore, this project was discribed and conducted. In this investigation, cuts 2 or 3 cm of fin tissue of  specimen of Cobia obtained from Sistan and Bluchestan, Hormozgan, Bushehr and Khuzestan water provinces, were collected. DNA was extracted by Phenol-chlorophorm method and produced PCR product in length of 1060 and 1450 base pair of two mitochondrial genes COI and NADH2. Using 13 cutting enzymes (4 enzymes were subscriber for both of genes), 205 base pair (from 2510 base pair, equal with %3.8 from gene regains) were directly investigated. But binding patterns of enzymatic digestion of PCR products of both COI and ND genes from electrophoresis were monomorph in all samples and no polymorphism was observed. This may be attributed to the unsuitable choice of COI and ND2 genes for showing of intra specific divergence. But in general non-existence of genetic diversity or noticeable decrease of that among individuals has been reported in regions were fish migration exist and they can freely move between two regions. Therefore, non-observation of polymorphism in the study area might be the case and indicates represents the area. On the other hand, some scientists believe that the distributions of populations in different regions are greatly affected by environmental and physical and ecological factors. Althoug Cobia is a migratory fish, but with regard to the fact that the environmental conditions are different (specially temperature and salinity) between east and west of Persian Gulf and Oman sea, there is a possibility that different genetic groups of this species exist in the regions. Of course It is clear that using more samples and enzymes from other genetically regions could produce better results. Since none of the two investigated genes didn’t show genetic divergence or polymorphism amongst the individuals of one region or between different regions, therefore, statistic analysis for estimating of haplotype diversity or nucleotide diversity and drawing of relationship tree among individuals using available softwares was not possible.

Genetic variation survey of intra-specific (Sepia pharaonis) in the Persian Gulf and the Oman Sea using PCR-RFLP

This research was conducted to identify Cuttlefish population (Sepia pharaonis) in The Persian Gulf and the Oman Sea using PCR-RFLP. Specimens were collected from )0 different stations. Bottom trawling method was used for sampling from different zones of the Persian Gulf and the Oman Sea, and finally specimens from S. Pharaonis were collected at each station . DNA was extracted by phenol—Coloroform method. One pair primer was designed based on 1As rRNA gene nucleotide sequences. The results obtained from 1 As rRNA gene RFLP, which was reproduced by PCR technique, were analyzed and utilized for study of diversity of the Cuttlefish population. PCR product with o pair base in length achieved for all specimens, which was subjected to enzymatic digestion by A restriction action enzymes: Alu I-Taq I-Mnl I-Rsa I-Hind III-Dra I-vu II and Hae II DNA bands patterns in all specimens digested by those enzymen showed similarity with no any polymorphism. From this result, it can be concluded that there is not any possibility to isolate different populations in the studied Cuttlefish species under exploitation of rRNA gene.

检测HIV-1载量的荧光实时定量PCR技术的建立及其应用

其它部委、高等院校基金;中国科学院基金

滤食性鲢、鳙肠含物PCR-DGGE指纹分析

以采自武汉东湖的滤食性鲢、鳙为对象,通过PCR-DGGE并结合序列分析对其肠道微生物及肠含物中残留的食物组分进行了探索研究。在所有个体中都能检测到不同的PCR-DGGE指纹谱带,其中包括肠道细菌在内的原核谱带较多,真核谱带相对较少;分析结果表明针对鲢、鳙肠含物这一特殊生境样品进行PCR-DGGE指纹分析是可行的。PCR-DGGE指纹结构及针对部分特定PCR-DGGE谱带的序列分析显示,从武汉东湖采集的鲢、鳙在食性上存在很大的重叠,并没有像基于常规食性分析文献报道的那样明显不同。基于肠含物DNA来进行鱼类食

原生动物单细胞PCR应用初探——以天蓝喇叭虫为例

以天蓝喇叭虫(Stentor coeruleus)为研究对象,探索了单细胞单基因PCR扩增及单细胞全基因组PCR扩增技术在原生动物中的应用.经过不断探索和优化条件后,试验取得了理想的结果.在40例单细胞单基因(SSU rDNA基因全序列)PCR的一次性扩增中,新鲜细胞和经过中性红染色的细胞都获得了100%的成功率,室温下酒精(95%)保存一周的细胞获得了82.5%的成功率.在单细胞全基因组PCR扩增中,采用高效高保真的phi29DNA聚合酶结合随机引物(Random Primer)进行扩增,获得了丰富且质

利用RAPD-PCR探讨天蓝喇叭虫生物群系分化

选择天蓝喇叭虫(Stentor coeruleus)作为研究对象,对武汉市南湖、月湖、关桥3个水体共5个样点天蓝喇叭虫(S.coeruleus)样本的总DNA进行随机扩增多态DNA聚类分析,以检测各个样本的遗传相似性和趋异程度,借以评估样本间的遗传变异度。结果如下:(1)从98条随机引物中筛选12条引物共扩增出89条大小为100～1500bp的清晰条带,平均每条引物扩增出7.4条片段。(2)用Rapdistance1.04分析显示,不同样点样本之间存在着一定的变异,其遗传距离在0.076～0.416之间。