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Ｄｅｔｅｃｔｉｏｎ ｏｆ Ｈｅｐａｔｉｔｉｓ Ａ ｖｉｒｕｓ ｉｎ ｓｈｅｌｌｆｉｓｈ ｂｙ ｒｖｅｒｓｅ ｔｒａｎｓｃｒｉｐｔｉｏｎ ＰＣＲTXＤｅｔｅｃｔｉｏｎｏｆＨｅｐａｔｉｔｉｓＡｖｉｒｕｓｉｎｓｈｅｌｌｆｉｓｈｂｙｒｅｖｅｒｓｅｔｒａｎｓｃｒ... 相似文献
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二温式RT-PCR检测对虾Taura综合征病毒的研究 总被引：16，自引：1，他引：15
设计了一对能扩增大小为231bp对虾Taura综合征病毒(TSV)某段基因的特异性引物,优化建立了能快速检测TSV的二温式RT-PCR。特异性和敏感性试验结果表明。二温式RT-PCR能对3个试验用TSV毒株的RNA进行扩增,并得到与预期大小一致的231bp的扩增产物,而其它3种对虾病原则无相同大小的特异性扩增产物出现;RT-PCR最低能检测到1pg的TSV RNA。应用RT-PCR对320份分别来自广西沿海不同对虾养殖场的对虾样品进行检测,结果共有85份检出TSV。说明TSV在广西沿海地区养殖对虾中已呈现出区域性流行趋势。同时也显示了RT-PCR在TSV临床检测中具有较高的实用性。 相似文献
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Diagnosis of iridovirus in large yellow croaker by PCR 总被引：1，自引：0，他引：1
A rapid and sensitive PCR-based method for the detection of the large yellow croaker iridovirus(LYCIV) is described,which involves the amplification of a 295 bp fragment of the LYCIV ATPase gene from DNA isolated from naturally infected fish spleen.Sequencing of LYCIV ATPase gene fragment showed it shared 100% nucleotide sequence homology with the corresponding region of the ATPase gene of red sea bream iridovirus(RSIV) and sea bass iridovirus(SBIV),suggesting that LYCIV was homologous with RSIV and SBIV at least in part of the gemone.The specificity and sensitivity of the PCR procedure were tested on the iridovirus-infected fishes,the expected fragment was detected from spleen DNA samples of infected fishes,whereas no fragments were amplified from healthy fish spleen DNA,white spot syndrome baculoviruses(WSBV) DNA and pseudorabies virus(PRV) DNA.Detection limit of this method was 10-7 ng positive plasmid DNA containing target sequence,equal to about 100 virions.In the infected experiment,first positive detection(1/4) appeared at Day 3 post-infection,all fish(4/4) tested positive at Day 7,however obvious symptoms were observed at Day 8,so LYCIV infection could be detected prior to the appearance of obvious symptoms.These results indicate that this PCR method could be used for early,rapid and specific detection of LYCIV infection. 相似文献
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Due to increasing population growth and anthropogenic pollution in the coastal zone, contamination of water and seafood with pathogens is probably responsible for the greatest number of human morbidities and mortalities worldwide. Hence, regular monitoring of waterborne pathogens is required to safeguard public health. Current techniques rely on the culturing of nonpathogenic indicator organisms (e.g. Escherichia coli or coliforms) for detection by inference. However, recent epidemiological evidence shows poor correlation between concentrations of E. coli/coliform and waterborne pathogens. Moreover, traditional methods are slow, not cost-effective, unable to distinguish harmful from benign strains, and fail to detect viable but nonculturable pathogens. The use of the polymerase chain reaction (PCR) has provided rapid and highly sensitive methods for the specific detection of pathogenic microorganisms. This paper briefly reviews some DNA-based technologies for waterborne pathogen detection, and describes our recent development of two new DNA-based technologies—quantitative multiplex PCR (Q-mPCR) and DNA microarray—that allow simultaneous and cost-effective detection and quantification of numerous pathogens in a single sample, which is superior to the culture methods currently in use. 相似文献
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锦鲤疱疹病毒实时荧光定量PCR检测方法的建立及应用 总被引：2，自引：0，他引：2
研究建立并完善了Taqman实时荧光定量PCR检测锦鲤疱疹病毒（KHV）的方法。首先通过选取KHV病毒TK基因的保守序列（GenBank AB182940）,利用PRIMER EXPRESS 2.0设计引物和探针。其中Taqman探针的5’端标记FAM,3’端标记TAMRA。然后利用梯度稀释的阳性样品克隆质粒进行定量PCR反应,制作标准曲线,得到病毒拷贝数与Ct值的关系为：Ct=-3.45 lgX＋42.73（相关系数R2=0.989）。通过试验检测得到实时荧光定量PCR对KHV的灵敏度为32个病毒核酸拷贝数,同时,设计的引物和探针对于鱼类细胞系和其它鱼类病毒没有扩增反应,表现出较好的特异性。实验结果表明,实时荧光定量PCR检测KHV的方法,具有特异性好、灵敏度高的特点,可以缩短检测时间,提高检测速度,非常适合口岸进出口检验检疫的要求。 相似文献
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Vibrio vulnificus is an opportunistic pathogen widely distributed in estuarine and coastal seawaters. In this study, a culture-free method was developed to rapid detection of V. vulnificus in all seasons, based on loop-mediated isothermal amplification (LAMP) targeting virulent-correlated gene (vcg). The new assay method allows differentiation between the virulent and non-virulent strain of V. vulnificus accurately. This method also allows effective detection of the pathogeny in winter when the bacterium lives in the viable but non-culturable (VBNC) state. A total of 30 costal seawater samples collected in all seasons were used for the evaluation of this method. The results show that the method is sensitive, accurate and convenient. 相似文献
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A robust damage detection method developed for offshore jacket platforms using modified artificial immune system algorithm 总被引：1，自引：0，他引：1
Mojtahedi A. Lotfollahi Yaghin M.A. Hassanzadeh Y. Abbasidoust F. Ettefagh M.M. Aminfar M.H. 《中国海洋工程》2012,26(3):379-395
Steel jacket-type platforms are the common kind of the offshore structures and health monitoring is an important issue in their safety assessment.In the present study,a new damage detection method is adopted for this kind of structures and inspected experimentally by use of a laboratory model.The method is investigated for developing the robust damage detection technique which is less sensitive to both measurement and analytical model uncertainties.For this purpose,incorporation of the artificial immune system with weighted attributes(AISWA) method into finite element(FE) model updating is proposed and compared with other methods for exploring its effectiveness in damage identification.Based on mimicking immune recognition,noise simulation and attributes weighting,the method offers important advantages and has high success rates.Therefore,it is proposed as a suitable method for the detection of the failures in the large civil engineering structures with complicated structural geometry,such as the considered case study. 相似文献
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A new method for the determination of dissolved double-stranded deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) in seawater was developed, evaluated and used to study the fates of these nucleic acids in marine ecosystems. These nucleic acids, which were pre-concentrated on a hydroxyapatite column, were determined fluorometrically by the use of ethidium bromide dye, which binds specifically to the double-stranded polynucleotide. No dissolved organic matter coexisting in the pre-concentrated sample solution interfered in the analysis of DNA and RNA. Column recoveries of DNA and RNA in a sample volume of up to 11 were 93% and 97%, respectively, and 90% of both at 51. The detection limits of DNA and RNA concentrated from a 51 sample by this fluorometric method were 0.6 and 1.1 μg l−1, respectively. The concentration of dissolved nucleic acids in the waters from Tokyo Bay and Sagami Bay showed great variation in space and time. DNA ranged from 1 to 32 μg l−1, and RNA from below the detection limit to 34 μg l−1. The total amount of phosphorus in nucleic acids was an important fraction (12.9 ± 8.2%) of the dissolved organic phosphorus (DOP) and showed a good correlation with DOP. 相似文献
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PCR‐based techniques to determine diet of the Australian sea lion (Neophoca cinerea): a comparison with morphological analysis 
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下载免费PDF全文 Kristian J. Peters Kathy Ophelkeller Herdina Nathan J. Bott Simon D. Goldsworthy 《Marine Ecology》2015,36(4):1428-1439
Comprehensive dietary information for the endangered Australian sea lion (Neophoca cinerea) is currently limited by the deficiency and poor quality of identifiable prey remains recovered from regurgitate and faeces and the difficulty of observing feeding in the wild. In this study, we investigated DNA‐based prey detection methods using conventional (end‐point) and quantitative real‐time PCR (qPCR) on faeces collected from two captive Australian sea lions fed experimental diets of whole teleost fish, squid and shark tissue. PCR prey detection methods using the mitochondrial cytochrome oxidase subunit I (COI) and 16S genes combined with clone sequencing were compared with prey identified using traditional hard part analysis. The molecular results indicated that prey DNA was degraded. However, prey amplification was successful by targeting short (71 bp) DNA fragments. Both conventional PCR and qPCR techniques significantly increased prey detection compared with analysis of hard parts. For both sea lions, the hard part analysis was constrained by sporadic and extremely low recovery of fish otoliths (<2%), and cephalopod beaks were not recovered from the 116 squid fed. Comparisons between PCR techniques indicated comparable prey detection frequencies for all species tested; however, the sensitivity and greater resolution of qPCR improved prey detection by ~25% in one sea lion fed the experimental squid and perch. The detection of squid DNA ≤ 6 day post‐ingestion by qPCR further exhibits the ability and potential of this method to detect low concentrations of infrequent or pulse prey. This study highlights the use of DNA‐based analysis to detect prey taxa in the absence of identifiable hard prey remains. 相似文献
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轮状病毒是引起婴幼儿急性腹泻的重要病原体之一,在水环境中存活时间长,导致人类感染的剂量低,因此寻求一种快速高效的定量检测海水中的轮状病毒方法势在必行。传统的细胞培养技术不但耗时,而且灵敏度低,现代分子生物学技术虽然克服了上述缺点,但是其感染性的信息无从获得。因此,本文建立了细胞培养结合实时定量PCR(ICC-qPCR)的方法,并于2010年冬季对渤海湾天津近岸重点海域表层海水中具有感染性的轮状病毒进行了定量调查。500 mL海水经浓缩,48 h细胞培养之后,用qPCR方法在7个海水样品中检测出3个样品具有感染性,其测定值范围为1.8×102copies～3.8×103copies,该海域感染性轮状病毒的含量为1～39 PFU/L。ICC-qPCR方法快速、灵敏,将细胞培养与实时定量PCR技术相结合,能对感染性病毒进行定量分析,因此有望在今后的水环境胃肠道病毒的监测中得到广泛应用。 相似文献
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Gene specific primers and DNA probe were designed based on the sequence of 18S rDNA cloned from the red tide alga Thalassiosira rotula. A real-time fluorescent quantitative PCR (RFQ-PCR) method was developed for quantitative detection of T.rotula. The RFQ-PCR assay data showed that the results obtained with the RFQ-PCR quite good agreement with those with the light microscope (LM) counting method, which suggested that the RFQ-PCR could be a useful method for red tide alga detection. 相似文献
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《Oceanologica Acta》1998,21(6):983-992
To improve the knowledge of the survival of enteric viruses in a marine environment, the influence of physico-chemical parameters (temperature, UV, salinity) on the survival of infectious poliovirus 1 and hepatitis A virus (HAV) in seawater was first studied, the influence of suspended solids (SS) on poliovirus adsorption and survival in seawater was then evaluated and the detection of rotavirus genome in environmental samples (shellfish, river water, treated wastewater) was finally investigated. The results show that temperature has a major impact on virus survival in seawater as the time necessary to inactivate 90 % of the virus (T90) is 671 days at 4 °C and only 25 days at 25 °C. Ultraviolet light (42 mW s cm−2) rapidly inactivates viruses but HAV is more resistant (T90 = 2.6 min) than the poliovirus 1 (T90 = 1.3 min). By contrast, seawater salinity has no effect on virus survival. In presence of SS, 90 % to 99.9 % of the viruses were adsorbed. This adsorption does not provide any protection for viruses with low SS concentrations (3 and 15 mg L−1) but a slight increase in virus survival was observed with a high SS concentration (500 mg L−1). Finally environmental sample analysis indicated that 20 % shellfish, about 40 % river water and 40 % treated wastewater tested positive for the rotavirus genome. 相似文献