斑点叉尾鮰肠败血症（ESC）PCR

由鮰爱德华氏细菌引起的肠败血症（ESC，又称爱德华氏病）是影响斑点叉尾鮰养殖最大的疾病。为快速和准确的诊断该疾病，本研究以NCBI公布的妇爱德华氏细菌eip基因（GeneBank登录号为AF037441）为模板序列，设计种特异性诊断引物，成功建立了用于斑点又尾鮰肠败血症病原菌的PCR检测方法。研究结果表明。该方法能够直接从发病斑点叉尾鮰脑、肝、脾和肾组织中检测到妇爱德华氏菌，检测的最低量为21个细菌；同时，该诊断方法与Ⅰ型荧光假单胞菌、点状产气单胞菌点状亚种、鳗弧菌、温和气单胞菌、肠型点状产气单胞菌、拄状黄杆菌、嗜水气单胞菌、河孤菌、豚鼠气单胞菌及海豚链球菌等常见水产病原菌无交又反应。临床样品检测证明。本研究所建立的PCR检测方法可以用于斑点叉尾鮰肠败血症的快速诊断。     

斑点叉尾肠败血症(ESC)PCR诊断方法的建立

由爱德华氏细菌引起的肠败血症(ESC,又称爱德华氏病)是影响斑点叉尾养殖最大的疾病。为快速和准确的诊断该疾病,本研究以NCBI公布的爱德华氏细菌eip基因(GeneBank登录号为AF037441)为模板序列,设计种特异性诊断引物,成功建立了用于斑点叉尾肠败血症病原菌的PCR检测方法。研究结果表明,该方法能够直接从发病斑点叉尾脑、肝、脾和肾组织中检测到爱德华氏菌,检测的最低量为21个细菌;同时,该诊断方法与I型荧光假单胞菌、点状产气单胞菌点状亚种、鳗弧菌、温和气单胞菌、肠型点状产气单胞菌、柱状黄杆菌、嗜水气单胞菌、河弧菌、豚鼠气单胞菌及海豚链球菌等常见水产病原菌无交叉反应。临床样品检测证明,本研究所建立的PCR检测方法可以用于斑点叉尾肠败血症的快速诊断。     

龟鲍曼不动杆菌和摩氏摩根菌双重PCR诊断方法建立

为建立龟源致病性鲍曼不动杆菌和摩氏摩根菌双重PCR诊断方法,应用鲍曼不动杆菌rpoB基因和摩氏摩根菌gyrB基因的特异性引物,对单重和双重PCR反应的退火温度、引物浓度、MgCl_2浓度、dNTP浓度等进行优化。试验结果显示,鲍曼不动杆菌rpoB基因和摩氏摩根菌gyrB基因引物分别进行PCR扩增后,均获得特异性条带,与预期结果一致;rpoB和gyrB基因的上、下游引物各为0.3μmol/L,退火温度为59.3℃,dNTP浓度为0.5 mmol/L,MgCl_2浓度为5 mmol/L时,PCR扩增效果最佳;特异性试验结果显示,本方法仅对摩氏摩根菌和鲍曼不动杆菌有扩增,对其他病原菌如肺炎克雷伯氏菌、维氏气单胞菌及恶臭假单胞菌等无扩增;灵敏性结果表明,本方法对鲍曼不动杆菌和摩氏摩根菌最低检测质量浓度分别为4.12×10~(-3) ng/μL和1.257×10~(-3) ng/μL。通过对临床病料样本的检测分析证明,本方法与单重PCR检测结果一致。本试验所建立的针对两种病原菌检测的双重PCR方法特异性强、灵敏度高,可为龟鳖类鲍曼不动杆菌和摩氏摩根菌的鉴别诊断和临床调查提供帮助。     

[鱼师]诺卡菌特异性PCR快速检测方法的建立

[鱼师]诺卡菌（Nocardia seriolae）是危害中国南方大口黑鲈（Micropterus salmoides）养殖业的主要病原之一，由于该菌在培养基上生长缓慢，对其分离鉴定造成诸多不便。文章根据[鱼师]诺卡菌16S-23S转录间隔区（ITS）序列设计特异性引物建立了[鱼师]诺卡菌特异性PCR快速检测方法。试验结果表明，利用设计的特异性PCR引物只能扩增出[鱼师]诺卡菌特异性片段，检出限为5pg模板DNA。在此基础上研制了PCR快速检测试剂盒并对[鱼师]诺卡菌人工感染的大口黑鲈组织进行了检测，结果显示该试剂盒能从未出现明显发病症状的大口黑鲈组织中检出阳性片段，阳性率为100％，比传统细菌分离鉴定方法更加灵敏、快速且高效，具有较好的应用前景。     

二温式聚合酶链反应鉴别诊断迟缓爱德华氏菌病

根据迟缓爱德华氏菌株23S rDNA基因,设计合成一对引物XZE15、XZE16,建立了二温式聚合酶链反应(PER)鉴别诊断迟缓爱德华氏菌株的技术.特异性试验表明,对3株迟钝爱德华氏菌株进行PCR扩增出与预期大小相一致的284 bp的特异性片段,但对3株钻鱼爱德华氏菌及其他对照鱼病病原体核酸模板的PCR扩增不出现任何条带;敏感性试验结果显示,该二温式PCR可以检测到10 Pg的迟钝爱德华氏菌DNA.     

用nested—PCR方法快速检测鲑鱼肾杆菌

描述了用嵌套式聚合酶链式反应(nested PCR)快速检测鲑鱼细菌性肾病病原鲑鱼肾杆菌的方法。以BKDR和BKDF为引物,扩增鲑肾杆菌编码57kDa主要可溶性蛋白基因中501bp的DNA片段,再用引物BKDR2和BKDF2扩增其中长度为314bp的DNA片段。用其它15种常见鱼类致病菌验证这两组引物的特异性,结果没有非特异性的DNA片段被扩增出来。用酚抽提法和煮沸加冻融的方法获得的细菌裂解产物,PCR检测的灵敏度均可达到1.8×103CFU·mL-1,用Nested PCR进一步扩增PCR扩增的产物,检测灵敏度可再提高100倍。检测鲑鱼肾杆菌菌悬液与鲟卵的混合物,结果表明,该方法能准确、可靠、快速地检测鲑肾杆菌。     

皱纹盘鲍幼鲍溃烂病病原菌的研究

从患有溃烂病的皱纹盘鲍幼鲍体上，分离到一种致病性细菌。经人工感染试验，证实该菌为皱纹盘鲍幼鲍溃烂病的病原菌。显微镜观察该细菌为革兰氏阴性，极生１～３根鞭毛，能运动的杆菌，大小为０．７～０．８×２．３～２．８ｕｍ。经细菌分类试验鉴定，该菌生长最适温度为２０～３０℃，最适ｐＨ值５．５～８．５。该菌在培养基Ｂ上产生蓝色的荧光素。能利用葡萄糖产酸不产气，果糖、Ｄ－半乳糖、甘露糖、木糖、阿拉伯糖、海藻糖发酵反应阳性，不能发酵乳糖、麦芽糖、鼠李糖、蔗糖，可分解山梨醇、肌醇、肌苷。过氧化氢酶、脲酶、氧化酶、及明胶液化反应阳性；能利用柠檬酸，不还原亚硝酸盐，不产生吲哚和硫化氢，ＭＲ和Ｖ．Ｐ．试验阴性。初步鉴定该病原菌为荧光假单胞杆菌。还进行了病原菌药敏试验，该菌对卡那霉素、呋喃唑酮、恶喹酸敏感，这些药物可作为防治该病的首选药。     

用PCR法快速筛选中国对虾含微卫星的重组阳性克隆

运用PCR在中国对虾的小片段基因组文库中快速筛选含有微卫星序列的重组阳性克隆。PCR中使用的引物为载体pGEM-3(zf( )上多克隆位点两侧的T7／Sp6启动子引物和根据微卫星核心重复序列设计的STR引物：STR1：5‘-ATATATATATAT－3‘：STR2：5’－TCTCTCTCTCTC－3’，直接使用含有重组克隆的菌液，在PCR反应前增加一段裂解细菌的高温。筛选出来的含有微卫星序列的重组阳性克隆经DNA测序验证。结果证明，采用PCR方法用菌液快速筛选含有微卫星序列的重组阳性克隆完全可行，而且具有简便、快速、高效、不涉及同位素操作等优点。     

罗非鱼海豚链球菌PCR检测方法的建立

近年来，海豚链球病已给我国及世界罗非鱼养殖带来了巨大的经济损失。为建立特异、敏感、快速的罗非鱼海豚链球菌PCR诊断方法，根据NCBI数据库已公布的海豚链球菌基因序列，设计合成种特异性引物CM1／CM2，进行海豚链球菌特异基因片段的PCR扩增试验、反应条件的优化及不同检测材料的比较。同时测试了方法的特异性和敏感性，对不同养殖鱼场的10份临床样品进行了检测。试验结果表明，引物CM1／CM2可以扩增到与预计大小相符的870bp海豚链球菌特异性基因片段；PCR反应与鮰爱德华氏细菌、Ⅰ型荧光假单胞菌、点状产气单胞菌点状亚种、鳗弧菌、温和气单胞菌、肠型点状产气单胞菌、柱状黄杆菌、嗜水气单胞菌和河弧菌水产常见病原菌均无交叉反应；能够检测的最低细菌数为20～30个海豚链球菌；同时，该PCR可以直接从病鱼的脑、肝脏、肾脏及脾脏组织扩增出特异性目的片段；临床菌株检测结果与基于菌株16srRNA基因序列系统进化分析结果一致。由于病鱼内脏组织总DNA、菌落及菌液可直接用于该PCR扩增，最大限度缩短了整个检测时间及降低了检测成本。方法的建立为致病性海豚链球菌检测提供了一种简便、快速的途径，具有较好的应用前景。     

一种简便、快速检测鸡毒支原体的PCR方法

鸡毒支原体（MG）是造成鸡慢性呼吸道疾病的主要病原菌。通常采用血清学方法诊断该病，由于存在非特异性交又反应，这给临床诊断带来困难。为解决MG快速诊断问题，该研究根据已发表的鸡毒支原体种特异性序列FMG-2合成一对引物（MG1，MG2），用PCR方法检测MG。结果表明，该PCR方法对MG能特异性扩增732bp目的片断，而对照菌（大肠杆菌、金黄色葡萄球菌等）却不能扩增出目的片段。PCR产物经测序，其序列与Genbank序列同源性为98．86％。用PCR方法检测60份感染样本，其阳性检出率为80％，而用传统的分离培养方法检出率仅为50％。     

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.     

Identification and detection of Pseudomonas plecoglossicida isolates with PCR primers targeting the gyrB region

Pseudomonas plecoglossicida is the agent of bacterial haemorrhagic ascites (BHA) in freshwater fish farming in Japan. To develop a rapid identification and detection method for P. plecoglossicida, a PCR amplification technique targeting the chromosomal DNA region coding the B subunit of the DNA gyrase (gyrB) was used. The nucleotide sequences of gyrB were determined in nine isolates of P. plecoglossicida and two other Pseudomonas species. On the basis of these determined sequences and the gyrB sequences of other Pseudomonas species or fish pathogenic bacteria deposited in international nucleotide sequence databases (GenBank/EMBL/DDBJ), PCR primers PL-G1F, PL-G1R, PL-G2F and PL-G2R were designed for specific amplification of the partial gyrB of P. plecoglossicida. The specificity of these primers in amplifying the gyrB of P. plecoglossicida was verified using selected strains of related bacterial species. The nested PCR technique was used to detect P. plecoglossicida from kidney and intestine of ayu. Primer pair PL-G1F and PL-G1R was used for the external PCR, and primer pair PL-G2F and PL-G2R for the internal PCR. Of 10 ayu juveniles, expected size PCR products were observed from intestine and kidney samples in one and two specimens, respectively. The PCR technique with primers based on the gyrB sequence is thus useful for the diagnosis of BHA.     

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.     

鱼类环境DNA研究中通用引物的筛选验证

为了筛选一个通用性和适用性良好的能够运用于环境DNA(eDNA)研究的鱼类引物,从相关文献中选取了鱼类线粒体基因组部分片段的5对引物,分别对线粒体D-loop区、16S rRNA基因、COI基因以及Cytb基因部分片段进行扩增。对千岛湖48种鱼类基因组DNA进行扩增后比较发现,引物16s和COI均可以取得良好的扩增效果,通用性优于其他几对引物。引物16s的扩增产物经凝胶电泳检测均出现明亮的目的条带,引物COI则有3种鱼的条带经凝胶电泳检测亮度较暗。利用上述引物对环境样品eDNA扩增时发现,只有16s和COI的引物具有良好的扩增效果,能够得到明显单一的亮带。对该两种引物的PCR产物克隆后测序比对发现,16s的PCR产物均为千岛湖常见鱼类物种的基因片段,COI的PCR产物则为细菌COI基因的部分片段。综上,我们认为引物16s在通用性和适用性上都更为适合作为鱼类群落结构eDNA研究的通用引物。     

凡纳滨对虾养殖池水中氨化细菌的鉴定及系统发育分析

2004年上海地区凡纳滨对虾养殖业遭受到了毁灭性的病害冲击。杨先乐等[1]认为2004年上海及周边地区凡纳滨对虾病害暴发流行的病原较为复杂,有可能是原来所发现的病毒,也可能是变异株或新的病原体,还可能是多种病原的混合感染。虽然目前凡纳滨对虾的疾病主要表现为病毒性疾病,但     

温和气单胞菌PCR快速诊断方法的建立

根据NCBI公布的温和气单胞菌(Aeromonas sobria)丝氨酸蛋白酶的基因序列,设计并选取一对能够快速而准确地检测温和气单胞菌的PCR引物,建立PCR快速检测体系,并对患病的鱼组织进行检测。研究结果表明,使用设计的引物能够扩增出与预计大小相符合的131 bp的特异性片段,具有较好的检测特异性,对靶标DNA的检测灵敏度为1.0 pg/20μL,对温和气单胞菌的检测灵敏度为50 cfu/20μL。样品的检测结果与实际发病情况一致,表明本研究成功建立了温和气单胞菌常规PCR检测体系,该体系可以用于温和气单胞菌的诊断和样品的检测。     

Identification of the Fish Species Processed to Fish Meal

Abstract

As a consequence of the BSE crisis in Europe, the components of meal produced from fish or warm-blooded animals are under stricter control. PCR-based techniques for species identification of fish meal have been developed and applied to meals produced from a single species each, including herring, capelin, anchovy, horse mackerel and blue whiting. DNA was extracted by means of the cationic detergent cetyltrimethylammonium bromide and a region of the mitochondrial cytochrome b gene was amplified using universal primers. The amplicon was further characterized by restriction fragment length polymorphism (RFLP) and single strand conformation polymorphism (SSCP) analysis. A number of fish meals could be differentiated due to their species-specific DNA patterns.     

Detection of Koi herpesvirus: impact of extraction method,primer set and DNA polymerase on the sensitivity of polymerase chain reaction examinations

Since virus isolation is seldom successful, KHV infection is commonly detected by PCR examination. A number of different PCR assays have been described in recent years. However, at present no commonly accepted PCR method is used amongst different laboratories. The aim of this study was to check if the examination of infected fish by different PCR methods yielded comparable results. We used tissue samples of three KHV‐infected koi, one KHV‐infected common carp, one KHV‐infected goldfish and one non‐infected common carp. DNA was extracted with DNAzol Reagent, High Pure PCR Template DNA Preparation Kit and QIAamp DNA Mini Kit. The DNA was tested by PCR with different combinations of published primer sets –KHV‐F and ‐R, KHV‐Gray‐2F and ‐2R and KHV‐TKf and ‐TKr – plus different DNA polymerases – a standard Taq DNA polymerase, a Platinum (hotstart) Taq DNA polymerase and a Platinum (hotstart) Pfx DNA polymerase with proofreading activity. The different extraction methods produced DNA solutions with different yields of DNA and different degrees of homogeneity. Also, the sensitivity of the PCR depended on the choice of the primer set and polymerase. Not all infected fish could be identified with all methods; there were large differences in the sensitivity between methods.     

快速鉴定四大家鱼早期资源种类的PCR方法

根据线粒体控制区序列设计了青鱼(Mylopharyngodon piceus)、草鱼(Ctenopharyngodon idellus)、鲢(Hypophthalmichthys molitrix)、鳙(Aristichthys nobilis)物种特异的PCR引物,通过对四大家鱼受精卵的PCR扩增,种间交叉扩增,相关种类早期资源的扩增以及未知种类鱼卵的鉴定发现,所设计的引物具有很高的种类特异性,对特定种DNA具有良好的扩增效果,但是不能进行交叉扩增,对四大家鱼外的其它种类也不能扩增。实验没有出现一例假阳性或假阴性结果,说明所设计引物的种类特异性高。该方法特别适合从鱼类早期资源大量样本中准确快速鉴定四大家鱼种类。     

Development of a novel PCR assay based on the 16S-23S rRNA internal transcribed spacer region for the detection of Lactococcus garvieae

Lactococcus garvieae is recognized as an emerging pathogen in fish. Several PCR‐based methods have been developed for the detection and identification of L. garvieae; however, the sensitivity of these methods is still in question regarding the discrimination of this organism from other closely related species. Two primers, ITSLg30F and ITSLg319R, were designed from the sequence in the 16S–23S internal transcribed spacer (ITS) region and used for the specific detection of L. garvieae. L. garvieae strains including fish isolates were positive by this method. In contrast, previously developed PCR methods showed false‐positive results with non‐L. garvieae species. Our results indicate that a PCR method using the newly designed ITS primer set provides a sensitive and efficient tool for the detection of L. garvieae in fish and aquaculture environments.