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白斑综合征病毒(WSSV)3种PCR检测方法的灵敏度比较 总被引：1，自引：0，他引：1
为了探讨不同PCR检测方法的灵敏度,分别利用TaqMan实时定量PCR、世界动物卫生组织(OIE)公布的巢式PCR引物(简称OIE)、黄海水产研究所种质资源与工程育种研究室(GB)设计的引物(简称GB)及2种巢式PCR对应的一步法PCR,对具有不同白斑综合征病毒(White Spot Syndrome Virus,WSSV)含量的中国明对虾(Fenneropenaeus chinensis)样品进行检测.结果显示,当使用已知病毒含量的标准品进行检测时,TaqMan实时定量PCR方法可以检测到l0个WSSV拷贝;OIE巢式PCR与GB巢式PCR方法分别可检测到104和103个WSSV拷贝;单独使用OIE巢式PCR的外引物和内引物扩增时,分别可检测到5×104和2.5×104个WSSV拷贝;单独使用GB巢式PCR的外引物和内引物进行一步法PCR扩增时,分别可检测到104和5×103个WSSV拷贝.使用上述PCR方法分别对44份未知WSSV含量的样品进行验证,定量PCR方法检测阳性率为84.09%,OIE巢式PCR与GB巢式PCR方法检测的阳性率分别为18.18%和27.27%;单独使用OIE巢式PCR的外引物和内引物扩增检测的阳性率均为15.91%;单独使用GB巢式PCR的外引物和内引物扩增检测的阳性率分别为18.18%和20.45%.根据以上结果,PCR方法检测WSSV的灵敏度由高到低依次为:定量PCR、巢式PCR、一步法PCR. 相似文献
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淋巴囊肿病毒(LCDV)、肿大细胞病毒属虹彩病毒(Mega)、赤点石斑鱼神经坏死病毒(RGNNV)、传染性造血器官坏死病毒(IHNV)、传染性胰脏坏死病毒(IPNV)、病毒性出血败血症病毒(VHSV)和传染性鲑鱼贫血症病毒(ISAV)是养殖鱼类主要的病毒性病原,危害巨大。为实现这7种病原的高通量、同步检测,本研究在分析这7种病毒基因序列的基础上,设计了9组扩增子拯救多重PCR(Arm-PCR)引物,并对扩增体系中的Taq酶、Mg2+、dNTP、Primer Mix浓度及退火温度等参数进行调整和优化,结合基因芯片检测技术,建立了同步检测7种鱼类病毒的Arm-PCR方法。优化后的Arm-PCR方法第一步PCR体系为：Taq酶(2.5 U/μl)1.0μl,10×PCR Buffer(含20 mmol/L的Mg2+)5μl,dNTP(各2.5 mmol/L)5μl,10×Primer Mix(各2μmol/L)9μl,模板1μl,ddH2O补足至50μl,退火温度为56℃。研究结果显示,该方法可以在1支反应管内对上述7种病毒的9个致病基因同步进行扩增和检测,检测灵敏度分别为101 copies/μl (RGNNV、VHSV、ISAV-NS、ISAV-MA)、102 copies/μl (LCDV、Mega、IHNV、IPNV)和103 copies/μl (大菱鲆红体病虹彩病毒,TRBIV)。该方法特异性强,与半滑舌鳎、石斑鱼、大菱鲆和牙鲆基因组DNA不产生交叉反应。本研究建立的可同步检测7种鱼类病毒的Arm-PCR方法具有高通量、高灵敏度、高准确性的优势,能有效提高工作效率,在鱼类病毒的筛查和流行病学调查领域有广泛的应用前景。 相似文献
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不同固定剂及固定时间对三角帆蚌DNA提取效果比较 总被引：2，自引：0，他引：2
以三角帆蚌(Hyriopsis cumingii)为对象,研究了无水乙醇,95%乙醇,4%多聚甲醛,福尔马林溶液,TE缓冲液5种固定剂不同固定时间对三角帆蚌斧足DNA提取质量及PCR扩增的影响。结果显示95%乙醇固定后提取的三角帆蚌基因组DNA降解少,DNA浓度高,OD260/OD2 8 0在1.8~2.0之间,OD260/OD230在1.5左右,表明95%乙醇固定后三角帆蚌基因组DNA的提取质量最好,再依次为无水乙醇、福尔马林溶液、4%多聚甲醛,DNA提取质量最差的是用TE固定的三角帆蚌。 相似文献
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低剂量对虾白斑综合征病毒粗提液对斑节对虾体内潜伏期病毒及血细胞的影响 总被引：1，自引：0，他引：1
利用含3×103、6×102、2×102copies/mL的对虾白斑综合征病毒(white spot syndromevirus,WSSV)粗提液和PBS液人工注射感染携带病毒量约1×105copies/g的斑节对虾,通过实时定量PCR法、显微镜观察法研究了感染后斑节对虾发病死亡率、肌肉内WSSV含量及总血淋巴细胞数和不同种类血细胞的比例组成变化。结果表明,3种浓度下斑节对虾累积死亡率分别为93.33%±2.89%、56.67%±5.77%和45.00%±5.00%,对照组没有发生死亡。注射3×103copies/mL的浓度组,在注射后30 min时,对虾肌肉内的病毒含量达到最低值(3.54×104copies/g),而后持续上升至48 h时达到最高值(3.12×108copies/g)后再次下降;另两个感染组,在注射后1 h时,肌肉内病毒含量达到最低值(分别为1.11×105、9.54×104copies/g),在感染后6 h时出现一个次高值(分别为1.58×105、1.11×105copies/g),而后又降低,在48 h时达到最高值(分别为1.48×107、5.46×106copies/g)后下降;对照组对虾肌肉内病毒含量没有出现显著变化。不同感染浓度组,斑节对虾总血淋巴细胞数波动幅度和出现峰值的时间不同,特别是注射3×103copies/mL组,对虾总血淋巴细胞数在12和48 h时出现极低值(分别为3.48×106、4.05×106/mL);3个感染组除个别时间点外,对虾总血淋巴细胞数的变化规律与肌肉中WSSV的扩增规律成负相关。4个处理组,半颗粒细胞比例在感染初期呈现明显上升的趋势,后期比例虽有起伏但均维持在较高水平;颗粒细胞和透明细胞比例在感染初期均呈现明显下降的趋势,中期均略有上升,但后期颗粒细胞比例显著低于初期,而透明细胞比例则与初期无显著性差异。 相似文献
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《渔业科学进展》2016,(2)
根据Gen Bank中公布的虾肝肠胞虫(Enterocytozoon hepatopenaei)(EHP)SSU r DNA序列设计1对特异性引物,建立并优化了EHP的SYBR Green I实时荧光定量PCR(q PCR)检测方法。结果显示,该方法在60℃的退火温度时扩增效果最好,产物的熔解曲线为1个单峰,构建的方法对8.3×101–8.3×108 copies/μl的EHP SSU r DNA片段的检测响应具有良好的线性关系,扩增产物阈值循环数(Ct)与模板起始量的对数[log(Sq)]的关系为Ct=–3.369 log(Sq)+39.364(R2=0.992),扩增效率为98.1%,检测灵敏度下限为8.3×101 copies/μl,在线性范围内具有良好的组内和组间重复性。对实际样品的检测表明该方法比已报道的套式PCR的检测灵敏度约高4倍。利用本方法对采集自江苏、海南和山东的3批凡纳滨对虾样品的肝胰腺组织DNA(Hp DNA)中的EHP SSU r DNA进行了q PCR检测,结果显示,EHP的载量指数与对虾生长速率呈负相关关系,肝胰腺中EHP载量在103 copies/(ng Hp DNA)时代表了较高的风险水平。本研究建立的q PCR方法具有特异、灵敏、快速、定量的优点,所建立的方法及检测数据可为EHP的防控提供技术参考。 相似文献
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高效液相色谱-串联质谱法检测3种渔业环境基质中的孔雀石绿及隐性孔雀石绿残留 总被引：1，自引：0，他引：1
为了准确评估渔业生态环境中的孔雀石绿(MG)及其代谢产物隐性孔雀石绿(LMG)的残留状况,建立了3种渔业环境基质(水体、底泥和底泥-水体混合物)中MG和LMG的高效液相色谱-串联质谱(HPLC-MS/MS)检测方法。通过考察不同前处理方法对不同基质中MG和LMG回收率的影响,优化仪器性能,确定了色谱和质谱分析条件。具体方法是:水体过滤后采用PRS固相萃取柱进行净化、富集;底泥采用乙腈-二氯甲烷(1∶1,v/v)提取,再旋蒸、富集;底泥-水体混合物用二氯甲烷提取后,采用PRS固相萃取柱净化、富集。采用Thermo C18色谱柱对待测物进行分离,以乙腈-0. 2%乙酸铵(1∶1,v/v)为流动相洗脱,电喷雾-多反应正离子监测模式监测,内标法定量。结果表明,3种不同基质中的MG和LMG在1～8 ng/m L范围内线性显著,其相关系数r~2值大于0. 999;加标回收率分别为90. 0%～104. 2%、79. 9%～90. 3%和74. 1%～86. 3%,相对标准偏差为3. 3%～5. 8%、4. 9%～8. 6%和3. 2%～8. 3%; MG的检出限(LOD,S/N=3)分别为0. 24 ng/L、0. 02μg/kg和0. 06μg/kg,定量限(LOQ,S/N=10)分别为0. 79 ng/L、0. 07μg/kg和0. 21μg/kg; LMG的检出限(LOD,S/N=3)分别为1. 14 ng/L、0. 17μg/kg和0. 12μg/kg,定量限(LOQ,S/N=10)分别为4. 72 ng/L、0. 56μg/kg和0. 39μg/kg。该法可应用于渔业环境中MG及LMG的定性定量检测,具有较好的实用性。 相似文献
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鲤疱疹病毒Ⅱ型TaqMan real-time PCR 检测方法的建立及应用 总被引：1，自引：1，他引：0
针对鲤疱疹病毒Ⅱ型(Cyprinid herpesvirus 2,CyHV-2)DNA解旋酶基因编码区序列设计特异性引物,利用PCR技术扩增出长度为1446 bp的基因编码区片段,克隆到pMD19T载体上,构建重组质粒.经PCR鉴定与测序分析确认正确后,以10倍梯度稀释重组质粒,作为标准模板进行TaqMan real-time PCR扩增,制作标准曲线,建立了鲤疱疹病毒Ⅱ型的荧光定量PCR检测方法.检测结果显示,标准曲线的相关系数(R2)达到0.9991,斜率为-3.412;对初始模板定量检测的范围为1×101～1×107 copies/μL;特异性试验结果表明,该方法可特异性地检测出鲤疱疹病毒Ⅱ型,而对大鲵虹彩病毒(GSIV)、锦鲤疱疹病毒(KHV)以及空白对照无检测信号.取江苏射阳和宝应两地疑似患病鲫组织核酸作为模板进行荧光定量PCR,结果表明反应体系中的病毒量分别为6.89×104 copies/μL和3.02×102 copies/μL.本研究建立的鲤疱疹病毒Ⅱ型TaqMan实时荧光定量PCR方法灵敏度高、特异性强,对因鲤疱疹病毒Ⅱ感染引起的养殖鲫造血器官坏死症的诊断与病毒病原定量检测有重要意义. 相似文献
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繁殖季节大鳍鳠血液学的初步研究 总被引：1，自引：0，他引：1
对嘉陵江繁殖季节内的大鳍鳠(Mystus macropterus)11项血液指标值进行了测定。其结果是:红细胞(RBC)的平均值为(1.246±0.208)×1012个/L;白细胞(WBC)的平均值为(2.643±0.696)×1010个/L;血红蛋白(Hb)的平均值为5.905±1.035 mg/100 mL;血球沉降率(ESR)的平均值为2.493±0.718 mm/h;红细胞比积(H t)的平均值为20.567%±4.974%;红细胞的脆性(Eof)在0.362%±0.035%氯化钠溶液中开始溶解,在0.211%±0.028%氯化钠溶液中完全溶解;红细胞的长径(CL)的平均值为12.356±1.579μm、短径(CS)的平均值为8.895±1.155μm;红细胞核的长径(NL)的平均值为4.523±0.607μm、短径(NS)的平均值为3.810±0.485μm;红细胞平均血红蛋白浓度(MCHC)的平均值为15.429%±5.154%;红细胞平均体积(MCV)的平均值为(1.671±0.453)×10-4μm3;红细胞的平均血红蛋白量(MCH)的平均值为(4.842±1.060)×10-8mg,且雌雄之间血液指标无显著性差异。此外,还将繁殖季节内大鳍鳠的血液指标与其它养殖鱼类进行了比较。 相似文献
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目的 合理选择病毒快速核酸提取方法。方法 分别采用2种商品化核酸快提取法——Cell-to-cDNA裂解液(A法)和基于磁珠核酸提取方法(InnuPREP MP Basic Kit A,B法),提取羊临床样品中的羊口疮病毒(ORFV)和羊痘病毒(CaPV)核酸，使用实时荧光定量核酸扩增检测系统(qPCR)和重组酶聚合酶扩增结合侧流层析技术(RPA-LFD)方法进行平行检测，并与常规核酸提取方法(宝生物病毒DNA/RNA提取试剂盒，C法)比较。结果 与C法相比，在A法和B法提取的46份疑似orf拭子中，qPCR的ORFV核酸检出率分别为70.8%和87.5%,RPA-LFD的检出率分别为69.5%和82.6%;在A法和B法提取的52份疑似CaP的拭子样品中，qPCR的CaPV核酸检出率分别为77%和90%,RPA-LFD的检出率分别为65.5%和89.7%。对组织样品分析发现：A法不能用于组织样品的核酸提取；在B法提取的52份疑似orf组织样品核酸中，与C法相比，qPCR的ORFV核酸检出率为93.7%,RPA-LFD的检出率93.3%;对B法提取的61份疑似CaP组织样品核酸中，qPC... 相似文献
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Association between the interannual variation in the oceanic environment and catch rates of bigeye tuna (Thunnus obesus) in the Atlantic Ocean 
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下载免费PDF全文 The environmental processes associated with variability in the catch rates of bigeye tuna in the Atlantic Ocean are largely unexplored. This study used generalized additive models (GAMs) fitted to Taiwanese longline fishery data from 1990 to 2009 and investigated the association between environmental variables and catch rates to identify the processes influencing bigeye tuna distribution in the Atlantic Ocean. The present findings reveal that the year (temporal factor), latitude and longitude (spatial factors), and major regular longline target species of albacore catches are significant for the standardization of bigeye tuna catch rates in the Atlantic Ocean. The standardized catch rates and distribution of bigeye tuna were found to be related to environmental and climatic variation. The model selection processes showed that the selected GAMs explained 70% of the cumulative deviance in the entire Atlantic Ocean. Regarding environmental factors, the depth of the 20 degree isotherm (D20) substantially contributed to the explained deviance; other important factors were sea surface temperature (SST) and sea surface height deviation (SSHD). The potential fishing grounds were observed with SSTs of 22–28°C, a D20 shallower than 150 m and negative SSHDs in the Atlantic Ocean. The higher predicted catch rates were increased in the positive northern tropical Atlantic and negative North Atlantic Oscillation events with a higher SST and shallow D20, suggesting that climatic oscillations affect the population abundance and distribution of bigeye tuna. 相似文献
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Liping Li Rui Wang Wanwen Liang Xi Gan Ting Huang Yan Huang Jian Li Yunliang Shi Honglin Luo Ming Chen 《广西水产科技》2014,(1)
Previously, we reported 10 PEGE types of 85 tilapia Streptococcus agalactiae(GBS), which shifted from Streptococcus iniae in China, by using PEGE method. Presently, larger and more representative tilapia GBS were isolated, for the ?rst time in China, to characterize their serotypes and genetic diversities more precisely than had done before. 168 GBS strains were distributed in ?ve provinces of China, in which Guangdong, Guangxi and Hainan were the major ones, holding36.9%(62/168), 37.5%(63/168) and 19.6%(33/168), respectively. Serotypes, Ia, Ib and III, were observed in these strains and the most predominant one was Ia(95.2%), which mainly distributed in Guangdong, Guangxi and Hainan. Ia initially occurred in 2009, it shoot up to 32.1% in 2010,but decreased to 16.1% in 2011 before went up to 45.2% in 2012. Ib sporadically occurred during2007–2011, III onlyoccurred in 2012. 14 different PFGE types, including 4 new types(N, O,P and Q), were observed, in which B, D, F and G were the predominant types, holding 83.9%(141/168) of the total GBS strains. Ia corresponded to 11 PFGE types(A–H, N–P), in which type D predominated(51%). Ib represented 3 genotypes(I, J and Q) and III harbored only 2genotypes(N and F). Type N and Fsynchronously presented in Ia and III. In summary, the genetic diversity of tilapia GBS varied by serotypes and changed with geographical locations and years.Although Iastillpredominated, new rareserotypeIII alreadyoccurred in China. 相似文献
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In this experiment, a feeding trial was performed to determine the effects of fructooligosaccharide (FOS) on growth performance, digestive enzyme activity and immune response of Japanese sea bass, Lateolabrax japonicus juveniles (initial weight 38.3 ± 0.5 g), and the fish were examined following feeding with six levels of FOS (0, 0.5, 1, 2, 4 and 6 g/kg) for 28 days. Significant enhancement of weight gain (WG) and specific growth rate (SGR) was found in fish fed 1 g/kg FOS incorporated diets (p < .05), while the feed conversion ratio (FCR) in the 1, 2 g/kg FOS groups reduced significantly compared with the control (p < .05). Besides, the crude lipid in the 4, 6 g/kg FOS groups increased significantly compared with the control (p < .05). On the other hand, the erepsin and lipase activities significantly elevated in intestine of fish fed 2 g/kg FOS (p < .05) and the lysozyme activity in serum of fish fed 2 g/kg FOS were significantly higher than that in the control (p < .05). Moreover, the alkaline phosphatase activities in serum of fish fed 0.5, 1, 2 g/kg FOS were significantly higher than in control (p < .05). Regression analysis showed that the relationships between dietary FOS levels and either SGR, FCR, erepsin or lysozyme activities were best expressed by regression equations, and the optimal inclusion levels are 1.37, 1.80, 3.06, 3.11, 1.93 and 1.80 g/kg for SGR, FCR, erepsin, lipase, lysozyme and total superoxide dismutase activities, respectively. Overall, this study revealed that FOS incorporated diets could beneficial for L. japonicus culture in terms of increasing the growth, digestion and immune activities. Under the present experimental condition, the optimal supplementary level of FOS in the diet of L. japonicus is 1–3 g/kg. 相似文献
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Iciar Martinez Amaya Velasco Ricardo Pérez-Martín Eskil Forås Michiaki Yamashita Carmen G. Sotelo 《Journal Of Aquatic Food Product Technology》2013,22(4):289-297
The purpose of this work was to evaluate the suitability of a method based on the presence of two restriction sites (for Hae III and Hindf I) in the mitochondrial NADH dehydrogenase subunit 5 (mt ND5) gene to identify Scomber species. The evaluation was performed on 144 reference and market samples by sequencing of the entire 505-bp fragment of the mt ND5 gene and of a 464-bp fragment of the Kocher fragment of the cytochrome b gene (mt Cytb). Sequence analysis of any of the two fragments allows the identification of each of the four Scomber species, but S. japonicus and S. colias had the same restriction sites at the ND5 amplicon and would not have been differentiated by this analysis. Similarly, loss of the Hae III site in some S. scombrus individuals would have misidentified them as not being Scomber. All the market products were correctly labeled except one acquired in Spain labeled as originating in the Atlantic and containing S. japonicus. 相似文献
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Growth hormone (GH) and reproduction: a review 总被引：5，自引：0，他引：5
Florence Le Gac Odile Blaise Alex Fostier Pierre-Yves Le Bail Maurice Loir Brigitte Mourot Claudine Weil 《Fish physiology and biochemistry》1993,11(1-6):219-232
Interaction between growth and reproduction occurs in many vertebrates and is particularly obvious at certain stages of the life cycle in fish. Endocrine interactions between the gonadotropic axis and the somatotropic axis are described, the potential role of GH being emphasised. A comparative analysis of these phenomena in mammals, amphibians and fish, suggests a specific role of GH in the physiology of puberty, gametogenesis and fertility. It also shows the original contribution made by studies on the fish model in this field of investigations. 相似文献
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Two gonadotropins (GtH; Qa and Qb) were purified by gel filtration and ion exchange chromatography from the pituitaries of Indian walking catfish (Clarias batrachus). The presence of GtH during purification was assessed by in vitro oocyte maturation and in vivo steroidogenic activity, and their identities were determined by elution profiles, molecular weight, biological activities and yield. The molecular weights of Qa and Qb were 37 and 42 kDa, respectively, and composed of distinct subunits (Qa: 20 and 14 kDa and Qb: 26 and 18 kDa). Polyclonal antibodies raised against Qa immunostained Qa, Qb and pituitary GtH cells. A competitive Qa‐ELISA was developed whose sensitivity was 6.25 ng mL?1 (1.25 ng well?1) with intra‐ (3.5%) and inter‐ (12.4%) assay coefficients of variation. Displacement curves parallel to the standard were obtained with plasma and pituitary extracts of catfish, Qb and carp GtHII. The assay was validated by measuring the plasma Qa levels after LHRH treatment and in relation to ovarian growth in the female catfish during different reproductive phases. Based on the results, Qa and Qb corresponded to fish LH and FSH respectively. The findings will increase the knowledge of the mechanisms controlling fish reproduction and identification of sensitive phases in fish in captivity for hormonal manipulation. 相似文献
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Controlled infection of Poecilia reticulata Peters (guppy) with Tetrahymena by immersion and intraperitoneal injection 下载免费PDF全文
下载免费PDF全文 G Sharon M Pimenta‐Leibowitz M C L Vilchis N Isakov D Zilberg 《Journal of fish diseases》2015,38(1):67-74
Tetrahymena is a protozoan parasite, which infects guppy, Poecilia reticulata Peters, and causes substantial economical losses in commercial farms worldwide. Studies of guppy infected by Tetrahymena require standardized infection protocols. The LD50 for Tetrahymena infection of guppies by intraperitoneal (IP) injection was calibrated, and the level obtained was 946 parasites per fish. Guppy infection with Tetrahymena by immersion, imitating the natural route of infection via the integument, was studied under normal or stress conditions. Exposure to cold and netting (CNI) and to cold only (CI) followed by immersion exposure to 10 000 Tetrahymena per mL resulted in 22.5% and 19.2% mortality, respectively, as compared to 14.2% and 10% in groups that were netted only (NI) or non‐stressed (I). Histopathology revealed that immersion infection resulted in a systemic infection. Lysozyme levels, measured 3 weeks after infection, were significantly higher in the CNI group (288 μg per mg protein) compared with CI‐, NI‐ and I‐treated groups (94.5, 64 and 62.3 μg mg?1, respectively). There was no evident parasite immobilization activity in body homogenates, suggesting no development of acquired immunity. Re‐infection by IP injection revealed no increase in protection in any of the treatment groups, mortality range of 56.3–75%, higher than in the non‐exposed control (40.6% mortality). 相似文献
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