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1.
Edwardsiella tarda has become one of the most important emerging pathogens in aquaculture industry.Therefore,a rapid,reproducible,and sensitive method for detection and quantification of this pathogen is needed urgently.To achieve this purpose,we developed a TaqMan-based real-time PCR assay for detection and quantification of E.tarda.The assay targets the hemolysin activator HlyB domain protein of E.tarda.Our optimized TaqMan assay is capable of detecting as little as 40 fg of genomic DNA per reaction.A standard curve was generated from the threshold cycle values(y) against log10(E.tarda genomic DNA concentration) as x.The intra-and inter-assay coefficient of variation(CV) values were less than 2.06% and 1.05% respectively,indicating that the assay had good reproducibility.This method is highly specific to E.tarda strains,as it shows no cross-reactivity to Edwardsiella ictaluri,a member of the same genus,or to nine other fish-pathogenic bacteria species belonging to three other genera.This sensitive and specific real-time PCR assay provides a valuable tool for diagnostic quantitation of E.tarda in clinical 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
3.
Yongxiang Yu Zheng Zhang Yingeng Wang Meijie Liao Bin Li Liangyi Xue 《中国海洋大学学报(英文版)》2017,16(5):831-839
Novel preservation condition without ultra-low temperature is needed for the study of pathogen in marine fishes. Freeze-drying is such a method usually used for preservation of terrigenous bacteria. However, studies using freeze-drying method to preserving marine microorganisms remain very limited. In this study, we optimized the composition of protectants during the freeze-drying of Edwardsiella tarda, a fish pathogen that causes systemic infection in marine fishes. We found that the optimal composition of protectant mixture contained trehalose (8.0%), skim milk (12.0%), sodium citrate (2.0%), serum (12.0%) and PVP (2.0%). Orthogonal and interaction analyses demonstrated the interaction between serum and skim milk or sodium citrate. The highest survival rate of E. tarda was observed when the concentration of NaCl was 10.0, 30.0 and between 5.0 and 10.0 g L?1 for preparing TSB medium, E. tarda suspension and protectant mixture, respectively. When E. tarda was frozen at ?80°C or ?40°C for 6 h, its survival rate was higher than that under other tested conditions. Under the optimized conditions, when the protectant mixture was used during freeze-drying process, the survival rate (79.63%–82.30%) of E. tarda was significantly higher than that obtained using single protectant. Scanning electron microscopy (SEM) image indicated that E. tarda was embedded in thick matrix with detectable aggregation. In sum, the protectant mixture may be used as a novel cryoprotective additive for E. tarda. 相似文献
4.
In recent years, Edwardsiella tarda has become one of the most deadly pathogens of Japanese flounder (Paralichthys olivaceus), causing serious annual losses in commercial production. In contrast to the rapid advances in the aquaculture of P. olivaceus, the study of E. tarda resistance-related markers has lagged behind, hindering the development of a disease-resistant strain. Thus, a marker-trait association analysis was initiated, combining bulked segregant analysis (BSA) and quantitative trait loci (QTL) mapping. Based on 180 microsatellite loci across all chromosomes, 106 individuals from the F1333 (♀: F0768 ×♂: F0915) (Nomenclature rule: F+year+family number) were used to detect simple sequence repeats (SSRs) and QTLs associated with E. tarda resistance. After a genomic scan, three markers (Scaffold 404-21589, Scaffold 404-21594 and Scaffold 270-13812) from the same linkage group (LG)-1 exhibited a significant difference between DNA, pooled/bulked from the resistant and susceptible groups (P <0.001). Therefore, 106 individuals were genotyped using all the SSR markers in LG1 by single marker analysis. Two different analytical models were then employed to detect SSR markers with different levels of significance in LG1, where 17 and 18 SSR markers were identified, respectively. Each model found three resistance-related QTLs by composite interval mapping (CIM). These six QTLs, designated qE1–6, explained 16.0%–89.5% of the phenotypic variance. Two of the QTLs, qE-2 and qE-4, were located at the 66.7 cM region, which was considered a major candidate region for E. tarda resistance. This study will provide valuable data for further investigations of E. tarda resistance genes and facilitate the selective breeding of disease-resistant Japanese flounder in the future. 相似文献
5.
In situ loop-mediated isothermal amplification (in situ LAMP) combines in situ hybridization and loop-mediated isothermal amplification (LAMP) techniques for chromosomal localization of DNA sequences. In situ LAMP is a method that is generally more specific and sensitive than conventional techniques such as fluorescence in situ hybridization (FISH), primed in situ labeling (PRINS), and cycling primed in situ labeling (C-PRINS). Here, we describe the development and application of in situ LAMP to identify the chromosomal localization of DNA sequences. To benchmark this technique, we successfully applied this technique to localize the major ribosomal RNA gene on the chromosomes of the Zhikong scallop (Chlamys farreri). 相似文献
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7.
The toxicities of 4 common endocrine-disrupting chemicals (EDCs), 17β-estradiol (E2), p,p′-dichlorodiphenyldichloro-ethylene (DDE), 4-nonylphenol (NP) and tributyltin (TBT), to sperm motility, fertilization rate, hatching rate and embryonic development of Barbel chub (Squaliobarbus curriculus) were investigated in this study. The duration of sperm motility was significantly shortened by exposure to the EDCs at the threshold concentrations of 10 ng L?1 for E2 and TBT, 1 μg L?1 for NP and 100 μg L?1 for DDE, respectively. The fertilization rate was substantially reduced by the EDCs at the lowest observable effect concentrations (LOECs) of 10 ng L?1 for E2 and TBT and 10 μg L?1 for DDE and NP, respectively. Of the tested properties of S. curriculus, larval deformity rate was most sensitive to EDC exposure and was significantly increased by DDE at the lowest experimental level of 0.1 μg L?1. Other EDCs increased the larval deformity rate at the LOECs of 1 ng L?1 for E2, 10 ng L?1 for TBT and 1 μg L?1 for NP, respectively. Despite their decreases with the increasing EDC concentrations, the hatching rate and larval survival rate of S. curriculus were not significantly affected by the exposure to EDCs. The results indicated that all the 4 EDCs affected significantly and negatively the early life stages of the freshwater fish S. curriculus. Overall, E2 and TBT were more toxic than NP and DDE, while DDE might be more toxic to larval deformity rate than to other measured parameters. Thus, the 4 EDCs showed potential negative influences on natural population dynamics of S. curriculus. Our findings provided valuable basic data for the ecological risk assessment of E2, DDE, NP and TBT. 相似文献
8.
Synechococcus sp.CC9311 is a marine cyanobacterium characterized by type IV chromatic acclimation(CA).A genetic transformation system was developed as a first step to elucidate the molecular mechanism of CA.The results show that Synechococcus sp.CC9311 cells were sensitive to four commonly used antibiotics:ampicillin,kanamycin,spectinomycin,and chloramphenicol.An integrative plasmid to disrupt the putative phycoerythrin lyase gene mpeV,using a kanamycin resistance gene as selectable marker,was constructed by recombinant polymerase chain reaction.The plasmid was then transformed into Synechococcus sp.CC9311 via electroporation.High transformation efficiency was achieved at a field strength of 2 kV/cm.DNA analysis showed that mpeV was fully disrupted following challenge of the transformants with a high concentration of kanamycin.In addition,the transformants that displayed poor growth on agar SN medium could be successfully plated on agarose SN medium. 相似文献
9.
Studies were conducted to determine the cause of the acute mortality of half-smooth tongue sole Cynoglossus semilaevis Günther juveniles in a fish farm in Jimo, Shandong Province, China, in June 2006. Gross signs of the diseased tongue sole
included several petechiae and ecchymoses on the body and fin necrosis and hemorrhagic lesion at the base of the fin. Bacteria
were isolated from kidney, liver and hemorrhagic lesions of the diseased tongue sole. Among14 strains, SJ060621 was proved
to be highly virulent to juvenile tongue sole with LD50 value of <1.0×105 colony forming units (CFU) mL−1, while the remaining 13 were avirulent. Among the 16 antibiotics tested, SJ060621 was sensitive to gentamicin and nitrofurantoin.
It was identified as Listonella anguillarum with conventional plate and tube tests in combination with API 20E analysis. 16S rRNA gene and partial HSP60 gene sequenceing
analysis revealed that the strain was highly homologous with L. anguillarum. Examination of the infected musculature by electron microscopy indicated numerous bacteria and lots of macrophages containing
phagocytosed bacteria. Histopathological investigations revealed severe necrotic degenerative changes in the infected organs.
Indirect immunofluorescence assay (IFA) was employed to detect the location of occurrence of bacteria, and bacteria were found
in aggregations in the inflammatory areas in musculature. 相似文献
10.
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
11.
Xiaolei Ma Kehou Pan Lin Zhang Baohua Zhu Guanpin Yang Xiangyang Zhang 《中国海洋大学学报(英文版)》2016,15(2):351-356
The gene ble from Streptoalloteichus hindustanus is widely used as a selective antibiotic marker. It can control the phleomycin resistance, and significantly increase the tolerance of hosts to zeocin. The unicellular marine microalga Nannochloropsis oculata is extremely sensitive to zeocin. We selected ble as the selective marker for the genetic transformation of N. oculata. After the algal cells at a density of 2×107 cells mL?1 was digested with 4% hemicellulase and 2% driselase for 1 h, the protoplasts accounted for 90% of the total. The ble was placed at the downstream of promoter HSP70A-RUBS2 isolated from Chlamydomonas reinhardtii, yielding a recombinant expression construct pMS188. The construct was transferred into the protoplasts through electroporation (1 kV, 15 μS). The transformed protoplasts were cultured in fresh f/2 liquid medium, and selected on solid f/2 medium supplemented with 500 ng mL?1 zeocin. The PCR result proved that ble existed in the transformants. Three transformants had been cultured for at least 5 generations without losing ble. Southern blotting analysis showed that the ble has been integrated into the genome of N. oculata. The ble will serve as a new dominant selective marker in genetic engineering N. oculata. 相似文献
12.
Saccharina japonica is a common macroalga in sublittoral communities of cold seawater environments,and consequently may have highly effi cient ribulose-1,5-bisphosphate carboxylase/oxygenase(Rubisco)activity for carbon assimilation.In our study,we cloned the full-length Rubisco gene from S.japonica(SJ-rbc).It contained an open reading frame for a large subunit gene(SJ-rbcL)of 1 467 bp,a small subunit gene(SJ-rbcS)of 420 bp,and a SJ-rbcL /S intergenic spacer of 269 bp.The deduced peptides of SJ-rbcL and SJ-rbcS were 488 and 139 amino acids with theoretical molecular weights and isoelectric points of 53.97 kDa,5.81 and 15.84 kDa,4.71,respectively.After induction with 1 mmol/L isopropyl-β-Dthiogalactopyranoside for 5 h and purifi cation by Ni 2+ affi nity chromatography,electrophoresis and western blot detection demonstrated successful expression of the 55 kDa SJ-rbcL protein.Real-time quantitative PCR showed that the mRNA levels of SJ-rbcL in gametophytes increased when transferred into normal growth conditions and exhibited diurnal variations: increased expression during the day but suppressed expression at night.This observation implied that Rubisco played a role in normal gametophytic growth and development.In juvenile sporophytes,mRNA levels of SJ-rbcL,carbonic anhydrase,Calvin-BensonBassham cycle-related enzyme,and chloroplast light-harvesting protein were remarkably increased under continuous light irradiance.Similarly,expression of these genes was up-regulated under blue light irradiance at 350 μmol/(m 2·s).Our results indicate that long-term white light and short-term blue light irradiance enhances juvenile sporophytic growth by synergistic effects of various photosynthetic elements. 相似文献
13.
Eutrophication, which is the enrichment of a water mass with inorganic and organic nutrients that support plant growth, is a key factor in stimulating phytoplankton growth. In this study, we determined the effects of various nitrogen sources, different nitrogen concentrations in the culture medium, and two culture methods on the growth of the green alga, Enteromorpha prolifera. The relationship between the specific growth rate of E. prolifera and NO3--N concentration was consistent with that estimated using the Monod equation (R2 = 0.9713, P < 0.01). In the NO3--N medium, the maximum specific growth rate was calculated to be 0.1634/d and the semi-saturation constant was calculated to be 16.86 μmol/L. Our results show that E. prolifera can effectively utilize NH4+-N, NO3--N, and NO2--N and urea-N in the range of 5 to 50 μmol/L. NH4+-N was preferentially assimilated by E. prolifera, and urea-N was favorable for long-term growth. 相似文献
14.
Ten actinomycete strains isolated from the Yellow Sea off China's coasts were identified as belonging to two genera by 16 S r DNA phylogenetic analysis: Streptomyces and Nocardiopsis. Six Streptomyces strains(MA10, 2SHXF01-3, MA35, MA05-2, MA05-2-1 and MA08-1) and one Nocardiopsis strain(MA03) were predicted to have the potential to produce aromatic polyketides based on the analysis of the KSα(ketoacyl-synthase) gene in the type II PKS(polyketides synthase) gene cluster. Four strains(MA03, MA01, MA10 and MA05-2) exhibited significant inhibitory effects on mycelia growth(inhibition rate 50%) and subsequent aflatoxin production(inhibition rate 75%) of the mutant aflatoxigenic Aspergillus parasiticus NFRI-95. The ethyl acetate extracts of the broth of these four strains displayed significant inhibitory effects on mycelia growth, and the IC50 values were calculated(MA03: 0.275 mg m L-1, MA01: 0.106 mg m L-1, MA10: 1.345 mg m L-1 and MA05-2: 1.362 mg m L-1). Five strains(2SHXF01-3, MA03, MA05-2, MA01 and MA08-1) were selected based on their high cytotoxic activities. The ethyl acetate extract of the Nocardiopsis strain MA03 was particularly noted for its high antitumor activity against human carcinomas of the cervix(He La), lung(A549), kidney(Caki-1) and liver(Hep G2)(IC50: 2.890, 1.981, 3.032 and 2.603 μg m L-1, respectively). The extract also remarkably inhibited colony formation of He La cells at an extremely low concentration(0.5 μg m L-1). This study highlights that marine-derived actinomycetes are a huge resource of compounds for the biological control of aflatoxin contamination and the development of novel drugs for human carcinomas. 相似文献
15.
Ya-bin Liu Ying Zhang Jiang-tao Fu Dong-mei Yu Xia-song Hu Xi-lai Li Zhao-xin Qi Shu-xia Li 《山地科学学报》2018,15(7):1532-1545
This study aims to assess the hydrological effects of four herbs and four shrubs planted in a selfestablished test area in Xining Basin of northeastern Qinghai-Tibet Plateau, China. The RainfallIntercepting Capability(RIC) of the herbs and shrubs was evaluated in rainfall interception experiment at the end of the third, fourth and fifth month of the growth period in 2007. The leaf transpiration rate and the effects of roots on promoting soil moisture evaporation in these plants were also assessed in transpiration experiment and root-soil composite system evaporation experiment in the five month's growth period. It is found that the RIC of the fourstudied herbs follows the order of E. repens, E. dahuricus, A. trachycaulum and L. secalinus; the RIC of the four shrubs follows the order of A. canescens, Z. xanthoxylon, C. korshinskii and N. tangutorum. The RIC of all the herbs is related linearly to their mean height and canopy area(R~2 ≥ 0.9160). The RIC of all the shrubs bears a logarithmic relationship with their mean height(R~2 ≥ 0.9164), but a linear one with their canopy area(R~2 ≥ 0.9356). Moreover, different species show different transpiration rates. Of the four herbs, E. repens has the highest transpiration rate of 1.07 mg/(m~2·s), and of the four shrubs, A. canescens has the highest transpiration rate(0.74 mg/(m~2·s)). The roots of all the herbs and shrubs can promote soil moisture evaporation. Of the four herbs, the evaporation rate of E. repens root-soil composite system is the highest(2.14%), and of the four shrubs,the root-soil composite system of A. canescens has the highest evaporation rate(1.41%). The evaporation rate of the root-soil composite system of E. dahuricus and Z. xanthoxylon bears a second-power linear relationship with evaporation time(R~2 ≥ 0.9924). The moisture content of all the eight root-soil composite systems decreases exponentially with evaporation time(R~2 ≥ 0.8434). The evaporation rate and moisture content of all the plants' root-soil composite systems increases logarithmically(R~2 ≥ 0.9606) and linearly(R~2 ≥ 0.9777) with root volume density. The findings of this study indicate that among the four herbs and four shrubs, E. repens and A. canescens possess the most effective hydrological effects in reducing the soil erosion and shallow landslide in this region. 相似文献
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17.
A new kinetic spectrophotometric method has been developed for the determination of iron (Ⅲ). The method is based on the catalytic effect of iron (Ⅲ) on the oxidation of weak acid brilliant blue dye (RAWL) by KIO4 in acid medium. The advantages of the proposed method are that it is sensitive, accurate, rapid, inexpensive, can be operated under room temperature and has a large determination concentration range compared to other techniques. The obtained optimum conditions are pH 3.15, RAWL (200 mgL-1) 5.00mL, Potassium periodate solution (0.01 molL-1) 0.30mL, phenanthroline (0.02molL-1) 1.00mL, reaction temperature 25℃ and reaction time 7 miu. With this method iron could quantitively be determined in the range 0.00-0.02 mgL-1, the detection limit being 4.10×10-10mL-1. The relative standard deviations (RSD) in five replicate determinations for 3 μgL-1 and 5μgL-1 iron (Ⅲ) are 3.1% and 1.9%, respectively. The method has been applied to the determination of iron (Ⅲ) in tap water samples and seawater samples (from the South China Sea), the recovery rates being 98.0% and 100.5%, respectively. 相似文献
18.
A 3-month feeding experiment was conducted in an in-door seawater system to investigate the effect of dietary vitamin E (Ve) on the sperm quality of turbot (Scophthalmus maximus). D-α-tocopherol acetate was supplemented to the basal (control) diet (65.14 mg kg?1 Ve) to obtain low and high levels of dietary Ve (244.60 mg kg?1, LVe; 721.60 mg kg?1, HVe). Compared with the control, sperm concentration was significantly increased in Ve-supplemented groups (LVe and HVe); while relative sperm volume and testis-somatic index were significantly increased in group HVe only. Sperm motility duration was significantly longer in group HVe than in the control, but no significant difference was observed in percent motility among groups. Sperm size, the uniformity of mitochondrial size, and the integrity of mitochondria cristae and plasma membrane were improved by dietary Ve, especially in HVe. The content of Ve in testis and liver as well as polyunsaturated fatty acids in sperm increased with dietary Ve. These results suggested that dietary Ve, especially at the high level (721.60 mg kg?1), significantly improved sperm concentration and motility duration and maintained normal sperm morphology of turbot. 相似文献
19.
Tingting Han Hongwei Ji Huixin Li He Cui Tian Song Xiaojuan Duan Qianlin Zhu Feng Cai Li Zhang 《中国海洋大学学报(英文版)》2017,16(3):455-460
Ion chromatography-ultra violet-hydride generation-Atomic Florescence Spectrometry was applied to detect 5 arsenic species in seafoods. The arsenic species studied include arsenobetaine(As B), arsenite(As(III)), dimethylarsinic acid(DMA), monomethylarsonic acid(MMA), and arsenate(As(V)), which were extracted from samples using 2% formic acid. Gradient elution using 33 mmol L~(-1) CH_3COONH_4 and 15 mmol L~(-1) Na_2CO_3 with 10 mL CH_3CH_2OH at pH 8.4 allowed the chromatographic separation of all the species on a Hamilton PRP-X100 anion-exchange column in less than 8 min. In this study, an ultrasound extraction method was used to extract arsenic species from seafood. The extraction efficiency was good and the recoveries from spiked samples were in the range of 72.6%–109%; the precision between sample replicates was higher than 3.6% for all determinations. The detection limits were 3.543 μg L~(-1) for As B, 0.4261 μg L~(-1) for As(III), 0.216 μg L~(-1) for DMA, 0.211 μg L~(-1) for MMA, and 0.709 μg L~(-1) for As(V), and the linear coefficients were greater than 0.999. We also developed an application of this method for the determination of arsenic species in bonito, Euphausia superba, and Enteromorpha with satisfactory results. Therefore, it was confirmed that this method was appropriate for the detection of arsenic species in seafood. 相似文献
20.
Junjie Yi Di Xu Xiaonan Zang Dingyang Yuan Bingran Zhao Li Tang Yanning Tan Xuecheng Zhang 《中国海洋大学学报(英文版)》2014,13(3):497-502
Arthrospira platensis is an economically important cyanobacterium; and it has been used widely in food and pharmaceutical industries. The phycocyanin(PC) from A. platensis is extremely valuable in medicine and molecular biology due to its antioxidation and anti-tumoring activity and applicability as fluorescence protein tag. In present study, two recombinant plasmids, one contained the phycocyanobilin(PCB)-producing genes(hox1 and pcyA) while the other contained the phycobiliprotein gene(cpcB) and the lyase gene(either cpcS/U or cpcT), were constructed and synchronically transferred into E. coli in order to test the the activities of relevant lyases for catalysing PCB addition to CpcB during synthesizing fluorescent PC holo-β-subunit(β-PC) of A. platensis. As was evidenced by the fluorescence emitted at a peak specific for PC, CpcB was successfully synthesized in E. coli, to which co-expressed PCBs attached though at a relatively low efficiency. The results showed that the attachment of PCBs to CpcB were carried out mainly by co-expressed CpcS/U but CpcB also showed some autocatalytic activity. Currently, no CpcT activity was detected in this E. coli expression system. Further studies will be conducted to improve the efficiency of fluorescent PC synthesis in E. coli. 相似文献