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1.
Edwardsiella tarda is a major pathogen in aquatic environments that can cause heavy economic losses. An improved method for quick and accurate detection of E. tarda by loop-mediated isothermal amplification (LAMP) with two additional loop primers was developed by targeting the EsrB gene (EsrB-LAMP). In this method, the Mg 2+ concentration, reaction temperature, and reaction time were optimized to 8 mmol/L, 61°C, and 40 min, respectively. The detection limit with the EsrB gene was as low as 10 copies, which is 100 times more sensitive than that of conventional polymerase chain reaction (PCR). The EsrB-LAMP assay was shown more sensitive and rapid than previously reported LAMP assays targeting the hemolysin gene (hemolysin-LAMP) for detection of E. tarda. The EsrB-LAMP was also highly specific to E. tarda and had no cross-reaction with 13 other strains of bacteria. The assay can be carried out in a simple heating device and the EsrB-LAMP products can be visually detected by adding fluorescent dye to the reaction mixture. Taken together, the improved EsrB-LAMP diagnostic protocol has the potential for detection of E. tarda from indoor and outdoor samples. 相似文献
2.
Vibrio anguillarum is an important bacterial pathogen of aquatic organisms and a significant problem in aquatic farming. The rapid detection
and identification of V. anguillarum, and other pathogens that infect marine organisms, is crucial to effective disease management. In this study, we developed
a loop-mediated amplification (LAMP) assay to detect V. anguillarum in an hour in a single tube without the need for thermal cycling. Conserved regions of the metalloproteinase (empA) gene of V. anguillarum served as the targets for primer design. A fragment of the empA gene was amplified at 65°C in the presence of the primer mixture and Bst DNA polymerase. In the optimized LAMP assay, 6.7 pg of V. anguillarum DNA could be detected. Six strains of V. anguillarum and 17 strains of non-V. anguillarum bacteria were used in this study to evaluate the species specificity of the primers. The six V. anguillarum strains gave a positive result in the LAMP assay. This method was also validated in V. anguillarum-infected fish. This LAMP method is more sensitive than PCR in the detection of V. anguillarum and shows good species specificity. The LAMP assay is therefore an effective method for the quick detection of V. anguillarum both in the laboratory and in the field. 相似文献
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Vibrio anguillarum is an important bacterial pathogen of aquatic organisms and a significant problem in aquatic farming.The rapid detection and identification of V.anguillarum,and other pathogens that infect marine organisms,is crucial to effective disease management.In this study,we developed a loop-mediated amplification (LAMP) assay to detect V.anguillarum in an hour in a single tube without the need for thermal cycling.Conserved regions of the metalloproteinase (empA) gene of V.anguillarum served as the targets for primer design.A fragment of the empA gene was amplified at 65°C in the presence of the primer mixture and Bst DNA polymerase.In the optimized LAMP assay,6.7 pg of V.anguillarum DNA could be detected.Six strains of V.anguillarum and 17 strains of non-V.anguillarum bacteria were used in this study to evaluate the species specificity of the primers.The six V.anguillarum strains gave a positive result in the LAMP assay.This method was also validated in V.anguillarum-infected fish.This LAMP method is more sensitive than PCR in the detection of V.anguillarum and shows good species specificity.The LAMP assay is therefore an effective method for the quick detection of V.anguillarum both in the laboratory and in the field. 相似文献
4.
Marine sponge Tedania anhelans distributes throughout the intertidal zone of Fujian, southeastern China, and is a potential source of natural bioactive products. The sponge harbors a large number of bacterial groups that have been identified using various techniques, including fluorescent in situ hybridization (FISH). Fractionation of dissociated sponge allowed isolation of 25 bacterial species. Based on 16S rRNA gene sequencing, phylogenetic analysis attributed most of these eubacteria to α-Proteobacteria, γ-Proteobacteria, Cytophaga/Flavobacterium/Bacteroidetes (CFB group), and the family Bacillaceae of Gram-positive bacteria. In sequence similarity, five putatively novel species were identified with less than 98% similarity to other strains in the NCBI database. Tests for antimicrobial activities were performed against Gram-positive bacteria, Gram-negative bacteria, fungi, antitumor indicators Escherichia coli 343/591 (with DNA repair deficiency), regular E. coli 343/636 (with different DNA repair capacity), and 10 bacterial isolates exhibited inhibitory bioactivities. Among these strains, three isolates were detected involving function gene NRPS-A domains, which were most closely related to the amino acid sequences of linear gramicidin synthetase and pyoverdine synthetase. These results contribute to our knowledge of the microbes associated with marine sponges and further reveal novel bacterial resources for the screening of bioactive marine natural products. 相似文献
5.
The family Sciaenidae is remarkable for its species richness and economic importance. However, the cytogenetic data available in this fish group are still limited, especially those obtained using fl uorescence in situ hybridization(FISH). In the present study, the chromosome characteristics of a sciaenid species, Argyrosomus amoyensis, were examined with several cytogenetic methods, including dual-FISH with 18 S and 5 S rDNA probes, and a self-genomic in situ hybridization procedure(Self-GISH). The karyotype of A. amoyensis comprised 2 n=48 acrocentric chromosomes. A single pair of nucleolar organizer regions(NORs) was located at the proximal position of chromosome 1, which was positive for silver nitrate impregnation(AgNO_3) staining and denaturation-propidium iodide(DPI) staining but negative for Giemsa staining and 4',6-diamidino-2-phenylindole(DAPI) staining, and was confi rmed by FISH with 18 S rDNA probes. The 5 S rDNA sites were located at the centromeric region of chromosome 3. Telomeric FISH signals were detected at all chromosome ends with dif ferent intensities, but internal telomeric sequences(ITSs) were not found. Self-GISH resulted in strong signals distributed at the centromeric regions of all chromosomes. C-banding revealed not only centromeric heterochromatin, but also heterochromatin that located on NORs, in interstitial and distal telomeric regions of specifi c chromosomes. These results suggest that the karyotype of Amoy croaker was relatively conserved and primitive. By comparison with the reported cytogenetic data of other sciaenids, it can be deduced that although the karyotypic macrostructure and chromosomal localization of 18 S rDNA are conserved, the distribution of 5 S rDNA varies dynamically among sciaenid species. Thus, the 5 S rDNA sites may have different evolutionary dynamics in relation to other chromosomal regions, and have the potential to be ef fective cytotaxonomic markers in Sciaenidae. 相似文献
6.
The dinoflagellate Alexandrium minutum is often associated with harmful algal blooms (HABs). This species consists of many strains that differ in their ability to produce toxins but have similar morphology, making identification difficult. In this study, species-specific rRNA probes were designed for whole-cell fluorescence in situ hybridization (FISH) to distinguish A. minutum from two phylogenetic clades. We acquired the complete SSU to LSU rDNA sequences (GenBank accession numbers JF906989-JF906999) of 11 Alexandrium strains and used these to design rRNA targeted oligonucleotide probes. Three ribotype-specific probes, M-GC-1, M-PC-2, and M-PC-3, were designed. The former is specific for the GC clade ("Global clade") of A. minutum, the majority of which have been found non-toxic, and the latter two are specific for the PSP (paralytic shellfish poisoning)-producing PC clade ("Pacific clade"). The specificity of these three probes was confirmed by FISH. All cells in observed fields of view were fluorescently labeled when probes and target species were incubated under optimized FISH conditions. However, the accessibility of rRNA molecules in ribosomes varied among the probe binding positions. Thus, there was variation in the distribution of positive signals in labeled cells within nucleolus and cytosol (M-GC-1, M-PC-3), or just nucleolus (M-PC-2). Our results provide a methodological basis for studying the biogeography and population dynamics of A. minutum, and providing an early warning of toxic HABs. 相似文献
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Standard FISH protocols using fluorochrome-labeled oligonucteotide probes have been successfully applied for in situ detection.However,optimized protocols of FISH for specific eukaryotes in marine environments are often not developed.This study optimized the conditions of fluorescence in situ hybridization (FISH) by using two polar isolated microalgae.The modified conditions were as follows: (1) 10 mg·mL-1 lysozyme solution pretreatment at 37℃ for 30 min;(2) the hybridization buffer including 20% formamide;(3) the hybridization condition was 47℃ for 6 h. The cells enumerated by FISH were compared with those enumerated by flow eytometry (FCM) and DAPI to confirm the cell loss and hybridization efficiency.The optimized protocol was also successfully applied to Arctic Ocean samples,which were found to be dominated by Micromonas sp.The modified protocol showed a high relative efficiency and could be successfully applied for the detection of specific microbial eukaryotes in environmental samples. 相似文献
9.
The microsatellite-enriched library was constructed using magnetic bead hybridization selection method, and the microsatellite DNA sequences were analyzed in Pacific abalone Haliotis discus hannai. Three hundred and fifty white colonies were screened using PCR-based technique, and 84 clones were identified to potentially contain microsatellite repeat motif. The 84 clones were sequenced, and 42 microsatellites and 4 minisatellites with a minimum of five repeats were found (13.1% of white colonies screened). Besides the motif of CA contained in the oligoprobe, we also found other 16 types of microsatellite repeats including a dinucleotide repeat, two tetranucleotide repeats, twelve pentanucleotide repeats and a hexanucleotide repeat. According to Weber(1990), the microsatellite sequences obtained could be categorized structurally into perfect repeats (73.3%), imperfect repeats(13.3%), and compound repeats (13.4%). Among the microsatellite repeats, relatively short arrays (＜ 20 repeats) were most abundant,accounting for 75.0%. The largest length of microsatellites was 48 repeats, and the average number of repeats was 13.4. The data on the composition and length distribution of microsatellites obtained in the present study can be useful for choosing the repeat motifs for microsatetlite isolation in other abalone species. 相似文献
10.
A full length amphioxus cDNA, encoding a novel phosducin-like protein (Amphi-PhLP),was identified for the first time from the gut cDNA library of Branchiostoma belched. It is comprised of 1 550 bp and an open reading frame (ORF) of 241 amino acids, with a predicted molecular mass of approximately 28 kDa. In situ hybridization histocbemistry revealed a tissue-specific expression pattern of Amphi-PhLP with the high levels in the ovary, and at a lower level in the hind gut and testis, hepatic caecum, gill, endostyle, and epipharyngeal groove, while it was absent in the muscle, neural tube and notochord. In the Chinese Hamster Ovary (CHO) cells transfected with the expression plasmid pEGFP-NIIAmphi-PhLP, the fusion protein was targeted in the cytoplasm of CHO cells, suggesting that Amphi-PhLP is a cytosolic protein. This work may provide a framework for further understanding of the physiological function of Amphi-PhLP in B. belcheri. 相似文献
11.
The microsatellite-enriched library was constructed using magnetic bead hybridization selection method, and the microsatellite DNA sequences were analyzed in Pacific abalone Haliotis discus hannai. Three hundred and fifty white colonies were screened using PCR-based technique, and 84 clones were identified to potentially contain microsatellite repeat motif. The 84 clones were sequenced, and 42 microsatellites and 4 minisatellites with a minimum of five repeats were found (13.1% of white colonies screened). Besides the motif of CA contained in the oligoprobe, we also found other 16 types of microsatellite repeats including a dinucleotide repeat, two tetranucleotide repeats, twelve pentanucleotide repeats and a hexanucleotide repeat. According to Weber(1990), the microsatellite sequences obtained could be categorized structurally into perfect repeats (73.3%), imperfect repeats(13.3%), and compound repeats (13.4%). Among the microsatellite repeats, relatively short arrays (＜ 20 repeats) were most abundant,accounting for 75.0%. The largest length of microsatellites was 48 repeats, and the average number of repeats was 13.4. The data on the composition and length distribution of microsatellites obtained in the present study can be useful for choosing the repeat motifs for microsatetlite isolation in other abalone species. 相似文献
12.
Sequences Characterization of Microsatellite DNA Sequences in Pacific Abalone(Haliotis discus hannai) 总被引：1，自引：0，他引：1
Kijima Akihiro 《中国海洋大学学报(英文版)》2007,(1)
1 Introduction Microsatellites or simple sequence repeats (SSRs) are tandemly repeated motifs of one to six bases found in all prokaryotic and eukaryotic genomes analysed to date (Zane et al., 2002). Due to their hyper-variable and co-dominant nature, relatively high abundance and random distribution in the genome, microsatellites are among the most efficient class of molecular markers. Such repeats display high polymorphism because of variation in repeat length and can be rapidly analysed t… 相似文献
13.
Sulfate-reducing bacteria (SRB), which obtain energy from dissimilatory sulfate reduction, play a vital role in the carbon and sulfur cycles. The dissimilatory sulfite reductase (Dsr), catalyzing the last step in the sulfate reduction pathway, has been found in all known SRB that have been tested so far. In this study, the diversity of SRB was investigated in the surface sediments from the adjacent area of Changjiang Estuary by PCR amplification, cloning and sequencing of the dissimilatory sulfite reductase beta subunit gene (dsrB). Based on dsrB clone libraries constructed in this study, diversified SRB were found, represented by 173 unique OTUs. Certain cloned sequences were associated with Desulfobacteraceae, Desulfobulbaceae, and a large fraction (60%) of novel sequences that have deeply branched groups in the dsrB tree, indicating that novel SRB inhabit the surface sediments. In addition, correlations of the SRB assemblages with environmental factors were analyzed by the linear model-based redundancy analysis (RDA). The result revealed that temperature, salinity and the content of TOC were most closely correlated with the SRB communities. More information on SRB community was obtained by applying the utility of UniFrac to published dsrB gene sequences from this study and other 9 different kinds of marine environments. The results demonstrated that there were highly similar SRB genotypes in the marine and estuarine sediments, and that geographic positions and environmental factors influenced the SRB community distribution. 相似文献
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In this study, the entire mitochondrial DNA （mtDNA） control region （CR） of Pholis fangi was amplified via polymerase chain reaction followed by direct sequencing. The length of the mtDNA CR consensus sequence of P. fangi was 853 bp in length. In accordance with the recognition sites as were previously reported in fish species, the mtDNA CR sequence of P. fangi can be divided into 3 domains, i.e., the extended terminal associated sequence （ETAS）, the central conserved sequence block （CSB）, and the CSB domain. In addition, the following structures were identified in the mtDNA CR sequence of P. fangi：2 ETASs in the ETAS domain （TAS and cTAS）, 6 CSBs in the central CSB domain （CSB-F to CSB-A）, and 3 CSBs in the CSB domain （CSB-1 to CSB-3）. These demonstrated that the structure of the mtDNA CR of P. fangi was substantially different from those of most other fish species. The mtDNA CR sequence of P. fangi contained one conserved region from 656 bp to 815 bp. Similar to most other fish species, P. fangi has no tandem repeat sequences in its mtDNA CR sequence. Phylogenetic analysis based on the complete mtDNA CR sequences showed that there were no genetic differences within P. fangi populations of the same geographical origin and between P. fangi populations of different geographical origins. 相似文献
17.
According to the known sequence of iron stress-induced gene (isiAB operon), we cloned its 1.5 kb fragment by PCR, and used this fragment as integration homologous fragment. After several steps of subcloning donor DNA into theisiAB fragment, a donor plasmid pZL which could be integrated into the chromosomal DNA ofSynechococcus sp. PCC7942 was constructed. In order to express the heterologous gene at a high level through the integration platform system, we constructed the donor DNA by the following steps. We cloned the strong promoter (240 bp) of heat shock genegroESL operon fromSynechococcus sp. PCC7942 by PCR. Then subcloned the multiple cloning sites (MCS),rbcS polyA into the downstream of thegroESL promoter. The kanamycin resistance gene, as the marker gene, was also subcloned into the donor DNA. Thus, in the donor plasmid pZL, the integration homologous fragment and several expression elements, such asgroESL promoter, MCS,rbcS polyA terminator and kanamycin resistance gene, were all included.
After naturally transformed and introduced the donor plasmid pZL intoSynechococcus sp. PCC7942, as in the pZL, the donor DNA sequence is flanked by two DNA fragments (0.4 kb and 0.7 kb) homologous to theisiAB fragment ofSynechococcus sp. PCC7942, the homologous DNA can recombine with the chromosomal DNA. After screening by kanamycin, the transformants which integrated the heterologous DNA were selected. The efficiency of transformation is about 1×10−6. By southern blot analysis, it was confirmed that the donor DNA had been integrated into the chromosomal DNA ofSynechococcus sp. PCC7942, located on the site of theisiAB gene, and can be replicated with the chromosomal DNA.
相似文献18.
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DU Guoying WU Feifei MAO Yunxiang GUO Shenghua XUE Hongfan and BI Guiqi 《中国海洋大学学报(英文版)》2014,13(1):97-103
An assessment with assistance of DNA barcoding was conducted on green macroalgae in coastal zone around Qingdao, China, during the period of April–December, 2011. Three markers were applied in molecular discrimination, including the plastid elongation factor tufA gene, the internal transcribed spacer (ITS) region of the ribosomal cistron and rubisco large subunit gene 3′ regions (rbcL-3P). DNA barcoding discriminated 8 species, excluding species of genus Cladophora and Bryopsis due to failures in amplification. We ascertained and corrected 4 species identified by morphological methods for effectively assisting the classification. The gene tufA presented more advantages as an appropriate DNA marker with the strongest amplification success rate and species discrimination power than the other two genes. The poorest sequencing success largely handicapped the application of ITS. Samples identified by tufA and rbcL as Ulva flexuosa were clustered into the clade of U. prolifera by ITS in the neighbor-joining tree. Confusion with discrimination of the complex of U. linza, U. procera and U. prolifera (as the LPP complex) still existed for the three DNA markers. Based on our results, rbcL is recommended as a preferred marker for assisting tufA to discriminate green macroalgae. In distinguishing green-tide-forming Ulva species, the free-floating sample collected from the green tide in 2011 was proved to be identical with U. prolifera in Yellow Sea for ITS and rbcL genes. This study presents a preliminary survey of green macroalgae distributed in the coastal area around Qingdao, and proves that DNA barcoding is a powerful tool for taxonomy of green macroalgae. 相似文献
20.
We isolated a strain of lymphocystis disease virus (LCDV) from Japanese flounder (Paralichthys olivaceus) cultured in northern China. Based on published sequences of major capsid protein (MCP) gene of LCDV-cn (GenBank: AF126405),
we designed two primer sets P1/P2 and P3/P4. We then used one-step or nested PCR and in-situ hybridization (ISH) to detect
LCDV and identify the target tissues or cells in infected Japanese flounder. The PCR products were positive in purified viral
supernatant, skin nodules, gut, gill, kidney, spleen, stomach, heart, and liver of Japanese flounder. We compared the DNA
sequence with 14 MCP nucleotide sequences from GenBank, including Megalocytivirus (OFIV and RSIV), Iridovirus (CzIV and WIV), Ranavirus (TFV and FV3), and Lymphocystivirus (8 LCDV). Based on the alignment, we confirmed the PCR product was from Lymphocystivirus (GenBank accession number DQ279090 (LCDV-HD)). Using ISH, we noted the presence of LCDV in the skin nodules, gut, gill, spleen,
stomach, and heart of spontaneously infected Japanese flounders. We successfully amplified LCDV fragments from Schlegel’s
black rockfish (Sebastes schlegeli Higendorf), redwing sea robin (Lepidotrigla microptera Günther) and turbot (Scophthalmus maximus) using the one-step and nested PCR, suggesting the target genes can be widely detected in fish using this method. 相似文献