创伤弧菌和哈氏弧菌双重PCR检测方法的建立及初步应用     

陈福艳  程光平  欧阳贤华  陈晓汉  梁万文  韦友传  黄婷  杨学明  陈明  王瑞  李丽萍  韦明利 《广东海洋大学学报》2013,(3)

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   

Potential use of dissolved cyanobacterial DNA for monitoring toxic Microcystis cyanobacteria in filtered water     

《Physics and Chemistry of the Earth》2013

Toxic and non-toxic Microcystis sp. are morphologically indistinguishable cyanobacteria that are increasingly posing health problems in fresh water systems by producing odours and/or toxins. Toxic Microcystis sp. produces toxicologically stable water soluble toxic compounds called microcystins (MCs) that have been associated with cases of aquatic life and wildlife poisoning and kills including some cases of human illnesses/deaths around the world. Thus, the need for rapid detection of toxic Microcystis sp. in surface water is imperatively a necessity for early mitigation purposes. Genomic DNA from potentially toxic Microcystis sp. comprises of ten microcystin synthetase (mcy) genes of which six major ones are directly involved in MCs biosynthesis. In Polymerase Chain Reaction (PCR) methodsmcy genes can be amplified from intracellular/extracellular genomic DNA using PCR primers. However, little is known about the limitations of sourcing genomic DNA templates from extracellular DNA dissolved in water. In this work, filtered water (0.45 μM) from a Microcystis infested Dam (South Africa) was re-filtered on 0.22 μM syringe filters followed by genomic DNA isolation and purification from micro-filtrates (9 mL). Six major mcy genes (mcyABCDEG) from the isolated DNA were amplified using newly designed as well as existing primers identified from literature. PCR products were separated by gel electrophoresis and visualized after staining with ethidium bromide. The limitation of using dissolved DNA for amplification of mcy genes was qualitatively studied by establishing the relationship between input DNA concentrations (10.0–0.001 ng/μL) and the formation of respective PCR products. The amplification of mcyA gene using new primers with as little as 0.001 ng/μL of DNA was possible. Other mcy gene sensitivities reached 0.1 ng/μL DNA dilution limits. These results demonstrated that with appropriately optimized PCR conditions the method can provide accurate cost-effective tools for rapid detection of toxic Microcystis sp. in water giving early information for water quality monitoring against MC producing cyanobacteria.  相似文献   

Reliable Procedure for Molecular Analysis of Airborne Microflora in Three Indoor Environments: An Office and Two Different Museum Contexts     

Carole Gaüzère  Marina Moletta‐Denat  Faisl Bousta  Stéphane Moularat  Geneviève Orial  Sébastien Ritoux  Jean‐Jacques Godon  Enric Robine 《洁净——土壤、空气、水》2013,41(3):226-234

Biological aerosols from air constitute a significant source of exposure to microorganisms in public places. Airborne microorganisms are involved in the development of certain respiratory symptoms, allergies, or infections among users and occupants. Various sampling instruments have commonly been used in aerobiology to collect bacteria and fungi suspended in the air. The objective of this study was to develop a reliable procedure for sampling in indoor public environments presenting different levels of occupancy, airborne bacteria and fungi to be subjected to molecular analysis (bacteria and fungi quantitative PCR, capillary electrophoresis single strand conformation polymorphism fingerprinting). Four different sampling devices were tested in situ in an office building (open‐plan type) and the sampling strategy chosen was tested in two museum contexts. In accordance with the drawbacks involved to our study (quantitative and qualitative aspects, cost, and overcrowding), cyclone device appeared to be most suitable. The results underline the effectiveness of this high‐volume aerosol sampling device for both qualitative and quantitative molecular analysis. Four in situ sampling collections were carried out in 1 day in the Louvre Museum to study quantitative and qualitative variations of airborne bacterial and fungal diversity. The quantitative results revealed a similar order of magnitude for the numbers of both bacteria and fungi. In the Louvre Museum, the samples yielded between 3.7 × 10⁴ and 4.1 × 10⁴ genome equivalent (GE) bacteria/m³ air and between 5.0 × 10⁴ and 5.9 × 10⁴ GE fungi/m³ air and in the Decorative Arts Museum between, 2.1 × 10⁴ and 2.5 × 10⁴ GE bacteria/m³ air and between 1.4 × 10⁴ and 1.7 × 10⁴ GE fungi/m³ air. The results also indicate that the dominant bacterial community displayed a stable structure over a short period of time whereas dominant eukaryotic airborne community appeared more variable.  相似文献   

CYP1A mRNA expression in redeye mullets (Liza haematocheila) from Bohai Bay, China     

An L  Hu J  Yang M  Zheng B  Wei A  Shang J  Zhao X 《Marine pollution bulletin》2011,62(4):718-725

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Major human Hepatitis A virus genotype in Hong Kong marine waters and detection by real-time PCR     

Yang N  Chu DL  Wong MM  Qi H  Wu RS  Kong RY 《Marine pollution bulletin》2011,62(12):2654-2658

Marine waters from seven sites around Hong Kong with varying levels of sewage pollution were analyzed for Hepatitis A virus (HAV) by PCR cloning and DNA sequencing of the highly variable VP1/2A junction of the HAV genome. Phylogenetic analysis of 10 PCR clones from each of the HAV-positive marine sites indicated that human HAV genotype IB is the most widely distributed type in Hong Kong waters. A sensitive and quantitative TaqMan-based PCR method targeting the 5′-noncoding region (5′-NCR) of HAV was used to quantify HAV particles in marine water samples along with the total Escherichiacoli counts being enumerated on TBX medium for comparison. Our results showed that no correlation of any significance between HAV and E. coli counts was observed which underscores the inadequacy in using E. coli as a sanitary standard to predict the levels of HAV in marine waters.  相似文献   

大菱鲆亲子鉴定的微卫星多重PCR技术建立及应用     

苗贵东  杜民  杨景峰  徐营  樊廷俊  陈松林 《中国海洋大学学报(自然科学版)》2011,(Z1):97-106

用8个微卫星标记组合建立了2个微卫星多重PCR体系,对大菱鲆7个人工选育家系进行了系谱认证、亲子鉴定和遗传多样性研究。2个多重PCR体系中8个微卫星位点共检测到等位基因49个,每个位点等位基因数为3~8个。根据已知亲本及子代基因型,成功推断出了3个缺失亲本的基因型。在双亲未知的情况下2个多重PCR的8个微卫星位点累积排除概率是96.58%,已知1个亲本时累积排除率为99.71%,亲子鉴定准确率为96.42%。采用70个个体进行双盲验证,利用UPGMA法对7个家系的70个体进行了聚类分析,同一家系95.71%的个体聚类分析结果与系谱来源一致。Cervus 2.0软件亲子鉴定结果表明亲子鉴定准确率为92.86%。2个多重PCR体系的建立为大菱鲆不同家系混养后的亲子鉴定、系谱分析和分子辅助家系管理提供了技术手段。  相似文献   

分子生物学技术在赤潮毒素分析监测中的应用   总被引：1，自引：0，他引：1  

冀秀玲  金桂文  程金平  王文华 《海洋科学进展》2004,22(1):107-113

有毒赤潮事件的频繁爆发,不仅对海洋生物及自然资源造成了极大的危害,还严重威胁到公众健康。因此,各国都在积极寻求快速灵敏的检测方法,加强对赤潮毒素的分析及监测。现代分子生物学技术的发展为新型快速检测方法的建立提供了可能。对利用分子生物学技术对赤潮毒素进行检测的几种方法——全细胞PCR法、DNA探针法及信使基因细胞受体法进行了讨论。这些新技术旨在检测出环境中的痕量赤潮毒素和有害生物,杜绝赤潮毒素或有毒藻进入到食物链,从而最大限度保护公众健康、水环境及自然资源。  相似文献   

两种体色三疣梭子蟹亲缘关系的PCR-SSCP分析     

迟大利  高焕  沈颂东  阎斌伦 《海洋通报(英文版)》2010,12(1):79-88

三疣梭子蟹作为重要的海洋经济动物,其常见体色为茶绿色。近年来在我国东部沿海海域海捕三疣梭子蟹中开始出现体色为紫色的一类梭子蟹,除体色不同外,二者表型特征并无显著差异。为确定两种体色三疣梭子蟹之间的关系,本文对两种体色三疣梭子蟹不同个体的线粒体COl和16srRNA基因进行了比较分析以判定体色的差异是否由于种属的差异引起的。通过对以上两个基因部分序列的SSCP和相关序列测定分析,发现在紫色和茶绿色群体中这两个线粒体保守序列基因的SSCP电泳条带图谱相似,序列分析表明COl和16srRNA基因在两种体色三疣梭子蟹群体中的核苷酸序列相似度分别为99．81％和99．91％。以上结果表明,紫色三疣梭子蟹并未发生亚种的分化,即紫色和茶绿色群体属于同一个种。此外,紫色梭子蟹群体的线粒体DNA序列在进化关系上较茶绿色群体更为保守。  相似文献   

海产鱼类中异尖线虫酶消化检测技术的研究与应用     

许旭  黄维义  隋建新  林洪  鲜均章  励建荣  曹立民 《中国海洋大学学报(自然科学版)》2010,40(3)

以3种海鱼(牙鲆、狭鳕、鲱鱼)为样本,针对海产品中主要的致病性寄生虫-异尖线虫,开展了酶消化检测技术的研究.根据鱼肉消化前后干物质的质量变化设计了胃蛋白酶消化效率的计算方法,并按照鱼肉:消化液＝1:10 (g/mL)的反应比例,分别确定了酶水解鱼肉的最佳条件为:初始pH值为1.1,温度为37 ℃,酶活力为8 U/mL左右;消化后所得的虫体采用多重PCR方法替代传统的形态学观察进行种属鉴定,从而初步建立了基于酶水解-多重PCR的异尖线虫的酶消化检测体系.实际样本检测表明,该技术可以较为准确、快速地用于海产鱼类中简单异尖线虫(Anisakis simplex)和伪地新线虫(Pseudoterranova decipiens)的确证性检测.  相似文献   

马氏珠母贝TRACP基因的克隆及组织表达     

郑哲  吴宣瑾  焦钰  黄荣莲  杜晓东 《广东海洋大学学报》2019,(3):24-29

【目的】克隆马氏珠母贝(Pinctada martensii)抗酒石酸酸性磷酸酶(Tartrate resistant acid phosphatase,TRACP)基因,分析该基因在不同组织中的表达模式。【方法】用RACE技术克隆得马氏珠母贝TRACP基因(PmTRACP),用实时荧光定量PCR分析该基因在外套膜、闭壳肌、足、性腺、珍珠囊、肝胰腺和鳃中的表达。【结果与结论】PmTRACP基因长度为2 034 bp,开放式阅读框972 bp,编码323个氨基酸,5′UTR长度为27 bp,3′UTR长度为1 035 bp。预测PmTRACP分子质量约为36.39 ku,理论等电点为5.97。该基因含有一个钙调神经磷酸酶样磷酸酯酶结构域。PmTRACP与其他物种TRACP的同源性为48%～65%,与长牡蛎(Crassostrea gigas)TRACP的同源性最高,同时D³²、D⁷⁰、Y⁷³、N¹⁰⁸、H²⁰³、H²¹²、H²³⁷、H²³⁹等8个氨基酸活性位点和N¹¹⁴糖基化位点在不同物种TRACP中高度保守。PmTRACP与长牡蛎TRACP的亲缘关系最近。PmTRACP在马氏珠母贝各个组织均有表达,且在肝胰腺和珍珠囊中高表达。  相似文献