共查询到19条相似文献，搜索用时 250 毫秒
1.
《广东海洋大学学报》2017,(3)
鰤鱼诺卡氏菌(Nocardia seriolae)为鱼类诺卡氏菌病的主要病原,绛红色诺卡氏菌(N.purpurea)是分离自土壤中一种无毒诺卡氏菌。以鰤鱼诺卡氏菌和绛红色诺卡氏菌为亲本进行原生质体融合,获取融合菌。经生长特征观察、革兰染色、16SrRNA测序分析,对融合菌进行初步鉴定,并通过斑马鱼模型评估融合菌毒力检测和免疫保护效果。结果表明,融合菌的菌落形态与亲本菌株不同,为诺卡氏菌属革兰阳性菌,融合菌的毒力较鰤鱼诺卡氏菌野生株降低70%,免疫保护率约为50%。 相似文献
2.
《广东海洋大学学报》2015,(6)
根据Gen Bank中类立克次氏体基因的保守序列,设计一对针对鱼类类立克次氏体PCR检测的特异性通用引物,通过对PCR扩增条件的优化,建立快速检测鱼类类立克次氏体的PCR方法,并用该方法对类立克次氏体阳性罗非鱼(Oreochromis nilotica)进行PCR扩增,结果得到与实验设计相符的390 bp的特异性扩增条带,而对健康罗非鱼、乌鳢(Ophiocephalus argus)、海鲈(Dicentrarchus labrax)、中华绒螯蟹(Eriocheir sinensis)及其他对照组的扩增结果为阴性。测序比对结果证实,该PCR方法检测结果准确,最低可检测出约1 pg的类立克次氏体质粒DNA;利用建立的PCR方法,对来自广东、云南、海南、天津、江苏、安徽、湖北等地的252份临床样品进行检测,共检出阳性样品30份,提示该PCR方法可用于类立克次氏体的临床快速检测。 相似文献
3.
用鲶爱德华氏菌兔抗血清作为一抗,碱性磷酸酶(AP)标记的羊抗兔IgG作为酶标二抗,建立黄颡鱼"红头病"病原菌—鲶爱德华氏菌的间接酶联免疫(ELISA)快速检测法,并优化检测条件。抗原最佳包被浓度为107/mL,一抗工作的最佳稀释度为1∶211,病原菌的检测灵敏度为105/mL,交叉反应实验证明该方法特异性强,与迟钝爱德华氏菌、弧菌等13种标准菌株无交叉。应用上述技术对人工感染发病鱼中分离的优势菌进行检测,结果表明阳性检出率为80%;对自然发病黄颡鱼体内分离获得的20株优势菌检测结果表明,12株菌为鲶爱德华氏菌。 相似文献
4.
卵形鲳鲹结节病病原的分离与鉴定 总被引：2，自引：0，他引：2
从湛江港、阳江闸坡海水网箱养殖的患结节病卵形鲳鲹(Trachinotus ovatus)中分离一株细菌,回归感染结果证明该菌是引起卵形鲳鲹结节病的病原,对卵形鲳鲹半致死浓度为45 g-1。该菌株呈革兰阳性,好氧,具有弱抗酸性,菌体呈短杆状或细长分枝状,过氧化氢酶阳性,氧化酶阴性,还原硝酸盐,不水解酪素、黄嘌呤、酪氨酸、淀粉、明胶,以柠檬酸盐为唯一碳源生长。对其16S rDNA序列作Blast分析,构建系统进化树。结果表明,该菌株与诺卡氏菌属的菌株亲缘关系最近,且与鰤鱼诺卡氏菌(Nocardia seriolea JCM 3360T)的16S rDNA序列同源性高达99%。综合患病鱼的症状及病原菌形态、生理生化特性及16S rDNA序列的结果,该菌被鉴定为鰤鱼诺卡氏菌(N.seriolea)。 相似文献
5.
《广东海洋大学学报》2015,(4)
根据Gen Bank中公布的对虾杆状病毒(Baculovirus penaei,BP)基因片段序列,设计1对特异性检测引物,建立快速检测凡纳滨对虾(Litopenaeus vannamei)对虾杆状病毒的PCR方法。用该方法对BP阳性虾进行PCR扩增,结果得到380 bp的特异性扩增条带,与实验设计相符,而对白斑综合征病毒(WSSV)、桃拉病毒(TSV)、传染性皮下及造血器官坏死病毒(IHHNV)阳性虾和健康虾的扩增结果为阴性。测序比对结果证实,该PCR方法检测结果准确,最低可检测出约1 pg的病毒DNA。利用建立的PCR方法对来自广东、广西、福建、海南和浙江的2 722份临床病料进行检测,共检出阳性病料133份,表明该PCR方法可用于对虾杆状病毒的临床快速检测。 相似文献
6.
以大豆病基因类似物(RGA)基因为对照,用RR大豆中的外源CaMV35S启动子、CP4-EPSPS引物,应用多重PCR方法,从RR大豆中扩增出预期大小的DNA片段。结果表明:将扩增产物回收后测序,经同源性分析扩增产物为CaMV35S启动子和CP4-EPSPS的一部分序列;多重PCR检测的灵敏度为0.08%;PCR检测过程中产生假阳性的原因是污染和非特异性扩增。 相似文献
7.
根据创伤弧菌(Vibrio vulnificus)的溶细胞毒素基因序列和哈氏弧菌(Vibrio harveyi)的toxR基因序列,分别设计并合成两对特异性引物,通过PCR反应条件优化,测试两种菌的特异性和敏感性,建立双重PCR方法,同时快速检测V.vulnificus和V.harveyi。结果表明:纯培养V.vulnificus和V.harveyi的检测灵敏度分别是12 cfu/mL和18 cfu/mL,与无乳链球菌、海豚链球菌、副溶血弧菌及美人发光杆菌无交叉反应;此PCR检测方法具有良好的特异性、敏感性,具快速、高效等优点,对细菌V.vulnificus和V.harveyi诊断与防治具有较好的临床应用性。 相似文献
8.
以大豆病基因类似物(ROA)基因为对照,用RR大豆中的外源CaMV35S启动子、CP4-EPSPS引物,应用多重PCR方法,从RR大豆中扩增出预期大小的DNA片段.结果表明:将扩增产物回收后测序,经同源性分析扩增产物为CaMV35S启动子和CP4-EPSPS的一部分序列;多重PCR检测的灵敏度为0.08%;PCR检测过程中产生假阳性的原因是污染和非特异性扩增. 相似文献
9.
【目的】研究广东湛江地区凡纳滨对虾(Litopenaeus vannamei)烂尾病主要病原及其药敏特征。【方法】从对虾病灶部位分离纯化细菌,用2株代表菌株对健康虾进行创伤浸浴感染试验,分析菌株形态特征、生理生化特征以及16S rRNA和HSP60基因序列,以哈维氏弧菌(Vibrio harveyi)和坎氏弧菌(V. campbelli)溶血素基因特异引物扩增菌株基因组DNA,通过纸片扩散法测试菌株对17种药物的敏感性。【结果】从患病亲虾和养殖对虾分别分离获得编号2018B22和2018MZ1的代表菌,两株菌16Sr RNA和HSP60基因序列均与哈维氏弧菌最相似,且均可被哈维氏弧菌溶血素基因引物特异扩增,表型特征亦与哈维氏弧菌相近;两株菌均可引起凡纳滨对虾烂尾病,其中菌株2018B22的致病力较2018MZ1更强,而后者的耐药谱更广。【结论】湛江地区凡纳滨对虾烂尾病主要病原为哈维氏弧菌。 相似文献
10.
采用聚丙烯酰胺凝胶电泳技术,从20对西大西洋笛鲷(Lutjanus campechanus)的微卫星引物中筛选适用于红鳍笛鲷(L.erythopterus)、勒氏笛鲷(L.russellii)、紫红笛鲷(L.argentimaculatus)和约氏笛鲷(L.johnii)基因组分析的微卫星引物,分析4种笛鲷的遗传多样性及系统发生。经反应条件的优化,从20对引物中筛选出17对可稳定扩增出特异片段的引物。通过测序证实扩增产物含微卫星位点后,以该17对引物对4个种的群体进行遗传多样性分析。其中16对可在约氏笛鲷、勒氏笛鲷、紫红笛鲷基因组中扩增出重复性好的特异性条带,分别有65%、65%、60%呈现出种内多态;15对可在红鳍笛鲷中扩增出重复性好的特异性条带,并且全部呈现出种内多态。四种笛鲷中,红鳍笛鲷的多态性最高,高度多态基因座占检测座位的66.67%;勒氏笛鲷的多态性最低,低度多态基因座占43.75%。获得3个种间特异性分子标记。用DS和DA两种方法计算4种笛鲷的遗传距离,构建了系统发生树。 相似文献
11.
YOU Feng WANG Wei XU Dongdong ZHU Xiangping NI Jing WU Zhihao XU Yongli WANG Xincheng ZHANG Peijun 《中国海洋湖沼学报》2009,27(2)
The hybrid between olive flounder Paralichthys olivaceus and stone flounder Kareius bicoloratus was produced by artificial insemination of olive flounder eggs with stone flounder Sperm. Sinistral and dextral are two types of hybrid progeny after metamorphosis. Karyotypes of both hybrid flounders are the same as those of the two parental species. Of the 22 loci examined from 12 allozymes,12 confirmed hybridization of the paternal and matemal loci in hybrids and no difference was found in allozyme patterns of sinistral and dextral hybrid fishes. RAPD patterns of these specimens were also studied with 38 primers selected from 104 tested. Among them, the PCR products of 30 primers showed hybridization of the paternal and maternal bands. Genetic variation between hybrids and their parental stocks was analyzed by RAPD using 10 of the above 38 primers. The average heterozygosity and genetic distance were calculated. The results suggested that the filial generation could inherit a little more genetic materials from paternal fish than that from maternal fish. 相似文献
12.
We isolated 4 Norwalk-like viruses (NLVs) contaminated oysters from 33 Chinese oysters collected from local commer-cial sources of Shandong Province. After amplification of the RNA-dependent RNA polymerase (RdRp) region of NLVs genomes with RT-PCR, the open reading frame 1 (ORF1) of the RdRp was sequenced and subjected to multiple-sequence alignment. The re-suits showed that NLVs in the four isolates belong to genogroup Ⅱ. The sequence comparison showed that the similarity between four Chinese oyster isolates were higher than 99.0%, which indicated that NLVs prevalent in close areas have high homogeneity in genome sequences. In addition, the most conserved sequences between diverse NLVs were used to design primers and TaqMan probes, then the real-time quantitative PCR assay was performed. According to the standard curve of GII NLVs, the original amounts (copies) of NLVs in positive patient's fecal isolate, positive Japanese oyster isolate, and the Chinese oyster isolate were 8.9×108, 1.25×108 and 4.7×101 respectively. The detecting limit of NLVs was 1×101 copies. This study will be helpful for routine diagnosis of NLVs pathogens in foods and thus for avoiding food poisoning in the future. 相似文献
13.
Hybrids between olive flounder Paralichthys olivaceus and stone flounder Kareius bicoloratus： karyotype, allozyme and RAPD analyses 总被引：2，自引：0，他引：2
The hybrid between olive flounder Paralichthys olivaceus and stone flounder Kareius bicoloratus was produced by artificial insemination of olive flounder eggs with stone flounder sperm. Sinistral and dextral are two types
of hybrid progeny after metamorphosis. Karyotypes of both hybrid flounders are the same as those of the two parental species.
Of the 22 loci examined from 12 allozymes, 12 confirmed hybridization of the paternal and maternal loci in hybrids and no
difference was found in allozyme patterns of sinistral and dextral hybrid fishes. RAPD patterns of these specimens were also
studied with 38 primers selected from 104 tested. Among them, the PCR products of 30 primers showed hybridization of the paternal
and maternal bands. Genetic variation between hybrids and their parental stocks was analyzed by RAPD using 10 of the above
38 primers. The average heterozygosity and genetic distance were calculated. The results suggested that the filial generation
could inherit a little more genetic materials from paternal fish than that from maternal fish.
Supported by National Natural Science Foundation of China (No. 30571445), National High-Technology Research and Development
Program (863 Program, No. 2006AA10A404), and Project from the Ministry of Science and Technology of China (No. 2006DKA30470-017) 相似文献
14.
We developed a species-specific PCR method to identify species among dehydrated products of 10 sea cucumber species. Ten reverse species-specific primers designed from the 16S rRNA gene, in combination with one forward universal primer, generated PCR fragments of ca. 270 bp length for each species. The specificity of the PCR assay was tested with DNA of samples of 21 sea cucumber species. Amplification was observed in specific species only. The species-specific PCR method we developed was successfully applied to authenticate species of commercial products of dehydrated sea cucumber, and was proven to be a useful, rapid, and low-cost technique to identify the origin of the sea cucumber product. 相似文献
15.
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
16.
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
17.
《中国海洋大学学报(英文版)》2015,(2)
Malachite green(MG), a dye, is an antifungal agent that has been used to treat and prevent fish diseases. It is metabolized into reduced leucomalachite green forms(LMG) that may reside in fish muscles for a long period, thus being harmful to human health. The aim of this study was to develop a competitive and direct enzyme-linked immunosorbent assay(ELISA) to detect MG and LMG specifically. The monoclonal antibody(m Ab) to LMG was generated using a hybridoma technique. The obtained m Ab showed good cross-reactivity(CR) to malachite green(MG), but not to crystal violet(CV) and Brilliant Green(BG). The m Ab was used to develop a fast detecting ELISA of MG and LMG in fish. By introducing the conjugation LMG-HRP, the detection capability was 0.37 ng m L-1 for MG and LMG. The mean recovery from spiked grass carp tissues ranged from 76.2% to 82.9% and the coefficients of variation varied between 1.8% and 7.5%. The stable and efficient monoclonal cell line obtained is a sustainable source of sensitive and specific antibody to MG and LMG. 相似文献
18.
Haiyang Yu Liming Jiang Wei Chen Xubo Wang Zhigang Wang Quanqi Zhang 《中国海洋大学学报(英文版)》2010,9(4):365-370
The expressed sequence tags (ESTs) of Japanese flounder, Paralichthys olivaceus, were selected from GenBank to identify simple sequence repeats (SSRs) or microsatellites. A bioinformatic analysis of 11111
ESTs identified 751 SSR-containing ESTs, including 440 dinucleotide, 254 trinucleotide, 53 tetranucleotide, 95 pentanucleotide
and 40 hexanucleotide microsatellites respectively. The CA/TG and GA/TC repeats were the most abundant microsatellites. AT-rich
types were predominant among trinucleotide and tetranucleotide microsatellites. PCR primers were designed to amplify 10 identified
microsatellites loci. The PCR results from eight pairs of primers showed polymorphisms in wild populations. In 30 wild individuals,
the mean observed and expected heterozygosities of these 8 polymorphic SSRs were 0.71 and 0.83 respectively and the average
PIC value was 0.8. These microsatellite markers should prove to be a useful addition to the microsatellite markers that are
now available for this species. 相似文献