双重PCR检测携带有tl和tdh基因的副溶血弧菌毒力菌株

在常规PCR技术的基础上优化条件,建立并完善双重PCR技术,并通过检测阳性对照和待检样品,进一步确定其检测的可行性.结果表明在常规PCR方法中为阳性的样品,包括阳性对照以及待检样品,在双重PCR方法中也呈现阳性,为阴性的样品在本方法中则也呈现阴性;能在血平板中出现溶血圈的在本法中也被印证含有tdh基因.由此证实本方法确实能对tl和tdh两种基因同时进行检测,成功地证明了同时检测tl和tdh两种基因的双重PCR方法的可行性.该法不但具有常规PCR的优点,而且还能节省耗材和时间,可用于检测水产食品以及临床样品中的副溶血弧菌的毒力菌株.     

广西凡纳滨对虾源副溶血弧菌3种毒力基因检测及tlh基因的蛋白表达

采用PCR技术检测分离自广西钦州、北海和防城港3市凡纳滨对虾样品中的67株副溶血弧菌(Vibrio parahaemolyticus)3种毒力基因tdh、trh和tlh的携带情况,并将tlh基因进行原核表达,SDS-PAGE和Western blotting鉴定分析。结果显示,67株副溶血弧菌均未扩增出tdh和trh基因,而tlh基因检出率为100.0%,所检副溶血弧菌的毒力基因型为tdh~-trh~-tlh~+。成功构建了原核表达质粒pET-28a-tlh,将其转化至大肠杆菌BL21 (DE3)感受态细胞后,经IPTG诱导表达,SDS-PAGE电泳检测得到大小约为53 kDa的产物,与预测值相符。经Western blotting鉴定,该重组表达产物能与抗6×His标签的单克隆抗体发生特异性反应。     

对虾养殖池副溶血弧菌的分离鉴定及其耐药特征、毒力基因分析

为了解对虾养殖池中副溶血弧菌(Vibrio parahaemolyticus)的耐药性和毒力基因的携带情况，2018年从山东4个地区的对虾养殖池收集分离副溶血弧菌，采用Kirby-Bauer纸片法检测其对12种抗生素的耐药性，用PCR方法检测其携带耐热直接溶血素基因(tdh)和耐热相关溶血素基因(trh)的情况。从对虾养殖池共分离副溶血弧菌50株。药敏实验结果显示，副溶血弧菌对庆大霉素、硫酸新霉素和氨苄西林的耐药情况最为严重，耐药率分别高达98%、90%和86%，对氟苯尼考、氯霉素、头孢他啶等敏感性较高，耐药率分别为10%、10%和20%。88%的菌株具有多重耐药性。毒力基因检测结果显示，所有菌株均不携带tdh基因，4%的菌株表现为trh阳性。本研究表明，对虾养殖水环境中的副溶血弧菌对抗生素的耐药性较为严重，应加强副溶血弧菌的病原学监测，在养殖过程中合理使用抗生素，以实现水产养殖业健康发展。     

凡纳滨对虾急性肝胰腺坏死病致病菌的分离鉴定、药敏特性及其组织病理学观察

为查明上海市奉贤区某对虾养殖场疑似患急性肝胰腺坏死病的凡纳滨对虾（Litopenaeus vannamei）的病原菌，本研究从患病凡纳滨对虾肝胰腺组织分离纯化到1株优势菌FHBX-1,并通过人工回归感染试验确定其致病性，利用VITEK-2全自动微生物鉴定仪和16S rRNA、热休克蛋白HSP60基因序列分析进行综合鉴定，并检测其毒力基因。同时采用K-B纸片扩散法检测病原菌的药物敏感性并通过石蜡切片观察患病凡纳滨对虾肝胰腺组织的病理特征。结果显示：菌株FHBX-1经生理生化特征测定、16S rRNA和热休克蛋白HSP60基因序列分析，综合鉴定为副溶血弧菌（Vibrio parahaemolyticus）,且携带了急性肝胰腺坏死病相关的毒力基因pirA^VP、pirB^VP,未携带副溶血弧菌临床主要毒力基因耐热直接溶血毒素tdh和相对耐热直接溶血毒素trh基因。人工回归感染试验结果显示，凡纳滨对虾在菌液浓度为1.0×10⁶ CFU/mL浸泡感染试验组，7 d内累计死亡率为80%,患病凡纳滨对虾具有肝胰腺颜色变白、萎缩变小，胃肠变...     

不同海洋弧菌中5类溶血素基因的分布及其与溶血活性和磷脂酶活性的相关性

用57株海洋弧菌,包括26株弧菌标准菌株、20株哈维氏弧菌、11株副溶血弧菌(从不同宿主和不同地理环境中分离得到),用PCR法合成5类相应的地高辛标记的溶血素基因探针,利用其进行Southern Blot,检测这5类溶血素基因在57株弧菌中的分布。结果显示,在57海洋株弧菌中,含有TDH、HlyA、TLH、δ-VPH和HLX溶血素基因的菌株分别为2株、2株、49株、3株和30株。另外,1株霍氏格里蒙菌(Grimontia hollisae)和1株副溶血弧菌(V.parahaemolyticus)中分别含有2个TDH溶血素基因。用鱼血平板和卵磷脂平板检测57株弧菌的溶血活性和磷脂酶活性,结果表明,弧菌溶血活性和磷脂酶活性与TLH溶血素基因具有显著相关性,与另外4类溶血素基因的关系不明显。     

洪湖碘泡虫PCR检测方法的建立与初步应用

为建立一种灵敏、特异的快速检测异育银鲫寄生洪湖碘泡虫(Myxobolus honghuensis)的方法,本研究根据洪湖碘泡虫ITS-5.8S r DNA基因序列筛选出一对特异性引物Mh F/R,建立PCR检测方法,对反应条件进行优化,并通过特异性试验、灵敏性试验与临床检测验证其可行性。结果显示,建立的PCR检测方法能特异性扩增洪湖碘泡虫相应的基因片段,长度为479 bp,而对试验中其他9种粘孢子虫的扩增结果均为阴性;最低能检测0.1 pg的虫体基因组DNA。通过临床样品检测,PCR方法比显微镜检测的检出率提高了19.5%。结果表明,该PCR方法特异、灵敏,适用于洪湖碘泡虫的快速检测。     

海水中副溶血弧菌的可视化LAMP快速检测方法

副溶血弧菌(Vibrio parahaemolyticus)是沿海地区常见的食源性致病菌之一,评估其在海洋环境中的存在状况对渔业生产与环境监测具有重要意义,但传统方法操作复杂或者设备繁重,往往不适合于现场快速检测。本实验采用环介导等温基因扩增法(loop-mediated isothermal amplification,LAMP),以tlh基因作为其标识基因,建立了海水中该菌的可视化快速检测方法。采用过滤法收集海水中的细菌,以细菌悬液为扩增模板,直接加入LAMP反应液中(免DNA提取与纯化步骤),以羟基萘酚蓝(hydroxynaphthol blue,HNB)为扩增结果指示剂,通过肉眼观察LAMP反应液颜色变化判断阴性、阳性结果。优化条件下,该方法反应约1 h可获得的最低检测限为167 CFU/m L。较之于传统PCR方法,所建立的方法分析周期短(从开始预处理样品到得出检测结果只需~2 h);灵敏度高(~10×PCR)。在验证实验中,应用该方法对青岛近岸海水中副溶血弧菌的检测,所获结果与经典培养方法和PCR方法结果一致。结果表明:该方法使用仪器简单,响应快,操作简易,可为现场快检海水中副溶血弧菌提供技术支持。     

双重PCR在创伤弧菌(Vibrio vulnificus)和副溶血弧菌(Vibrio parahaemolytious)快速检测中的应用

建立双重PCR方法检测海产品中的创伤弧菌(Vbrio vulnificus)和副溶血弧菌(Vibrio parahaemolytious)。针对创伤弧菌和副溶血弧菌的gyrB基因的不同位点设计引物,前者扩增片断的大小为968bp,后者扩增片断的大小为284bp。共对三种海产品进行了创伤弧菌和副溶血弧菌的检测,结果在部分海产品中能特异性地检测到目标菌株。该方法快速,灵敏度高,特异性强。为海产品的创伤弧菌和副溶血弧菌的检测提供了有效的方法。     

鳗源创伤弧菌的鉴定与血清型分析

本研究对分离自发病鳗鲡的疑似创伤弧菌进行鉴定及血清型分析,经生化鉴定,从发病鳗鲡中得到7株创伤弧菌;设计了创伤弧菌溶血素基因(vlly)特异性引物,并对分离菌进行了PCR检测,创伤弧菌均可扩增出溶血素基因352 bp的目标片段,而非创伤弧菌未扩增出相应的片段;制备了创伤弧菌抗O抗原血清,凝集反应显示所得到的鳗源创伤弧菌抗O抗原血清不能与人源创伤弧菌1.1758发生凝集反应,菌株FJ03-X2抗O抗原血清可与大部分的鳗源创伤弧菌发生凝集反应。以上研究结果表明,基于溶血素基因设计的PCR引物具有良好的特异性,可用于创伤弧菌检测,鳗源创伤弧菌血清型不同于其他来源的创伤弧菌,菌株FJ03-X2的O抗原具有良好的免疫原性,可作为创伤弧菌疫苗研发的候选菌株。     

鲤疱疹病毒2型和3型三重PCR检测方法的建立与应用

鲤疱疹病毒2型和3型主要感染鲫(金)鱼(Carassius auratus)、鲤鱼(Cyprinus carpio)和锦鲤(Cyprinus carpio haematopterus)及其杂交种,具有高度的传染性和致病性,对养殖鲤科鱼类危害严重。为了建立快捷、高效且能同时检测这2种类型鲤疱疹病毒的检测技术,本研究根据鲤疱疹病毒的DNA聚合酶基因(2、3型)、解旋酶基因(2型)和胸苷激酶基因(3型)的保守序列,设计了3对特异性引物,通过对三重PCR的反应条件进行优化,建立起鲤疱疹病毒2型和3型三重PCR检测方法,并运用该方法对实验室保存的鲫鱼和鲤鱼组织样品进行检测。结果显示,该三重PCR检测方法仅在鲤疱疹病毒阳性样品中扩增出3条特异性条带,表现出良好的特异性;以克隆的目的基因质粒为模板,进行10倍梯度稀释,该检测方法能检出的极限值为2.85×102 copies/μL,表现出较高的灵敏性;运用该方法检测122份鲫鱼和60份鲤鱼样品,其结果与运用国家标准(GB/T 36194-2018)和行业标准(SC/T 7212.1-2011)方法检测的结果一致。本研究表明,构建的三重PCR检测方法不仅具有较高的准确性和灵敏度,还能同时检测2种类型的鲤疱疹病毒,有效提高检测效率。     

Occurrence of Listonella anguillarum in seed production environments of Japanese flounder Paralichthys olivaceus (Temminck et Schlegel)

The present study was undertaken to investigate the distribution of Listonella anguillarum in the rearing water, fish and diets (rotifers) of Japanese flounder (Paralichthys olivaceus). A total of 793 isolates were obtained from the seed production environment of Japanese flounder and 175 out of them were identified as L. anguillarum by biochemical characterization, polymerase chain reaction (PCR) detection for VAH1 haemolysin gene and phylogenetic analysis of 16S ribosomal deoxyribonucleic acids (rDNA) sequences. These results strongly suggested that L. anguillarum is rapidly and accurately identified by the combination of incubation on thiosulphate–citrate–bile salt–sucrose agar at 35°C overnight and PCR detection for the VAH1 haemolysin gene. All flounder specimens and all rotifer samples harboured L. anguillarum at high densities of 6.9 × 10³–6.3 × 10⁵ colony forming units (CFU) g^?1 and 1.5 × 10⁴–2.3 × 10⁶ CFU g^?1, respectively, while as low as 5.0 × 10⁰–2.0 × 10¹ CFU mL^?1 of L. aguillarum were detected in only two of 11 seawater samples, even though no vibriosis occurred in larval and juvenile flounder of tanks. This fact strongly suggests that L. anguillarum is an inhabitant in the seed production environments of Japanese flounder.     

Identification of Vibrio parahaemolyticus in Seafood by Multiplex PCR

ABSTRACT

Vibrio parahaemolyticus is a human pathogen frequently found in seafood. Once the seafood is contaminated by V. parahaemolyticus, it can become a vehicle for foodborne illness. The conventional culture methods for detection of V. parahaemolyticus are time-consuming and cannot differentiate pathogenic strains from nonpathogenic ones. In this study, a multiplex polymerase chain reaction (PCR) technique was investigated for detecting tdh, chiA, and toxR of V. parahaemolyticus. The sensitivity of the multiplex PCR was determined by testing 28 strains of V. parahaemolyticus, 15 non-V. parahaemolyticus strains, and fresh seafood spiked with cells of V. parahaemolyticus. All the strains were analyzed for production of thermostable direct hemolysin (TDH) and chitinase. This study showed that both the chiA and toxR are excellent markers for detecting V. parahaemolyticus strains, and a multiplex PCR targeting chiA and tdh genes can be applied to simultaneously detect environmental and pathogenic V. parahaemolyticus.     

Insights into the virulence‐related genes of Edwardsiella tarda isolated from turbot in Europe: genetic homogeneity and evidence for vibrioferrin production

Edwardsiella tarda has long been known as a pathogen that causes severe economic losses in aquaculture industry. Insights gained on E. tarda pathogenesis may prove useful in the development of new methods for the treatment of infections as well as preventive measures against future outbreaks. In this report, we have established the correlation between the presence of virulence genes, related with three aspects typically involved in bacterial pathogenesis (chondroitinase activity, quorum sensing and siderophore‐mediated ferric uptake systems), in the genome of E. tarda strains isolated from turbot in Europe and their phenotypic traits. A total of 8 genes were tested by PCR for their presence in 73 E. tarda isolates. High homogeneity was observed in the presence/absence pattern of all the strains. Positive results in the amplification of virulence‐related genes were correlated with the detection of chondroitinase activity in agar plates, in vivo AHL production during fish infection and determination of type of siderophore produced by E. tarda. To the best of our knowledge, this is the first study carried out with European strains on potential virulence factors. Furthermore, we demonstrated for the first time that E. tarda produces the siderophore vibrioferrin.     

龟致病性维氏气单胞菌和鲍曼不动杆菌双重PCR检测方法的建立

应用2对特异性引物,通过对反应条件和体系的优化,成功建立了能快速检测维氏气单胞菌和鲍曼不动杆菌两种病原的双重PCR方法。研究结果表明,该方法特异性强,仅对龟鳖维氏气单胞菌和鲍曼不动杆菌有扩增,而对其他龟鳖常见病原无扩增;该方法对维氏气单胞菌最低检测核酸质量浓度为1.75×10-3 ng/μL,对鲍曼不动杆菌最低检测核酸质量浓度为3.96×10-3 ng/μL,检出率较传统检测方法高。该研究所建立的双重PCR方法灵敏度高、特异性好,可为临床龟鳖维氏气单胞菌和鲍曼不动杆菌感染的诊断和流行病学调查等提供帮助。     

白斑综合征病毒(WSSV)3种PCR检测方法的灵敏度比较

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.     

Rapid detection and identification of viral and bacterial fish pathogens using a DNA array-based multiplex assay

Fish diseases can be caused by a variety of diverse organisms, including bacteria, fungi, viruses and protozoa, and pose a universal threat to the ornamental fish industry and aquaculture. The lack of rapid, accurate and reliable means by which fish pathogens can be detected and identified has been one of the main limitations in fish pathogen diagnosis and fish disease management and has consequently stimulated the search for alternative diagnostic techniques. Here, we describe a method based on multiplex and broad-range PCR amplification combined with DNA array hybridization for the simultaneous detection and identification of all cyprinid herpesviruses (CyHV-1, CyHV-2 and CyHV-3) and some of the most important fish pathogenic Flavobacterium species, including F. branchiophilum, F. columnare and F. psychrophilum. For virus identification, the DNA polymerase and helicase genes were targeted. For bacterial identification, the ribosomal RNA gene was used. The developed methodology permitted 100% specificity for the identification of the target species. Detection sensitivity was equivalent to 10 viral genomes or less than a picogram of bacterial DNA. The utility and power of the array for sensitive pathogen detection and identification in complex samples such as infected tissue is demonstrated in this study.     

Complete mitochondrial DNA sequence of the Japanese anchovy Engraulis japonicus

ABSTRACT: The complete nucleotide sequence of the mitochondrial genome for the Japanese anchovy Engraulis japonicus (Teleostei: Clupeiformes) was determined. The entire genome was purified by gene amplification using the long polymerase chain reaction (PCR) technique, and products were subsequently used as templates for PCR with 56 fish-versatile primers that amplify contiguous, overlapping segments of the entire genome. Direct sequencing of the PCR products demonstrated that the genome (16 675 base pairs [bp]) contained the same 37 mitochondrial genes (two ribosomal RNA, 22 transfer RNA and 13 protein-coding genes) as those found in other vertebrates, with the gene order being identical to that in typical vertebrates. A major non-coding region between the tRNA^Pro and tRNA^Phe genes (1024 bp) was considered to be the control (D-loop) region, as it has several conservative blocks characteristic to this region.     

Rapid identification of Streptococcus iniae by specific PCR assay utilizing genetic markers in ITS rDNA

The 16S-23S intergenic spacers (ITS) of ribosomal DNA from ten independent isolates of Streptococcus iniae and one reference strain ATCC29178 were sequenced, aligned and used to design a polymerase chain reaction (PCR) primer set for rapid and specific detection and identification of S. iniae. This primer set amplified a 377-bp DNA fragment specifically from S. iniae, but not from other common bacterial pathogens of fish or from non-fish pathogens. The PCR conditions were optimized to allow detection of the organism from agar, broth culture or infected fish tissue. The sensitivity of the PCR assay was established by the detection of DNA as low as 0.02 ng or as few as 10 CFU bacterial cells. The establishment of the specific PCR assay provides a useful tool for the identification and diagnosis of fish infection with S. iniae.     

蛙病毒PCR检测方法的建立与比较分析

为了进一步丰富蛙虹彩病毒检测方法，实验针对蛙病毒3型(frog virus 3,FV3)核衣壳蛋白(major capsid protein,MCP)基因保守区设计一对特异性引物，并选取另外2种蛙病毒PCR检测方法进行对比实验，通过反应体系的优化、反应特异性和敏感性实验，建立了一种针对FV3的PCR检测方法。结果显示，该方法最低检测限可达1.2个拷贝数的病毒粒子，与神经坏死病毒、虾血细胞虹彩病毒、大口黑鲈虹彩病毒、锦鲤疱疹病毒、鲤浮肿病毒、传染性脾肾坏死病毒、加州鲈弹状病毒等常见水产动物致病毒株无交叉反应。临床样品检测表明，该方法所获得的检测结果与另外2种方法一致，结果可靠。本研究所建立的FV3 PCR检测方法，具有简便、快速、敏感度好、特异性高、低成本等特点，可用于FV3蛙病毒的快速诊断和分子流行病学调研。     

我国渔用疫苗的研制

定量（quantitive PCR）是用PCR技术评估样本中靶基因的分子数，实现对核酸信息的量化分析及比较。目前主要有外标法和内标法两大类。其中，其中外标法中主要有PCR产物的直接定量法，极限稀释法等；内参标法中主要有竞争性和非竞争性PCR等。