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1.
哈维氏弧菌胶体金免疫层析试纸条的制备 总被引：1，自引：0，他引：1
用18~20nm胶体金标记抗哈维氏弧菌Vibrio harveyi抗体并制备金标垫,分别将羊抗兔Ig G和纯化后的抗哈维氏弧菌抗体包被于NC膜上作为质控线和检测线,组装制作了哈维氏弧菌胶体金免疫层析试纸条,优化了该试纸条的制作条件,测试了其性能。结果表明:所制备的哈维氏弧菌免疫层析试纸条具有较好的特异性,与费尼斯弧菌Vibrio furnissii、维氏气单胞菌Aeromonas veronii、美人鱼发光杆菌Photobacterium damselae、类志贺邻单胞菌Plesiomonas Shigelloides、鱼肠道弧菌Vibrio ichthyoenteri、鳗利斯顿氏菌Listonella anguillarum、迟缓爱德华氏菌Edwardsiella tarda、海豚链球菌Streptococcus iniae、嗜水气单胞菌Aeromonas Hydrophila等9种常见水产病原菌无明显交叉反应,检测灵敏度为3×105CFU/m L,5~10min即可得出检测结果,具有快速、简便、特异性强和适应基层推广应用等优点,可用于养殖鱼类病原哈维氏弧菌的现场检测。 相似文献
2.
本研究检测了分离自发病大菱鲆、半滑舌鳎及鲤鱼的22株病原鳗弧菌(Vibrio anguillarum)毒力相关基因的携带情况,并建立了病原鳗弧菌的分子生物学检测方法。以PCR方法检测8个毒力相关基因的分布,结果显示,22株病原鳗弧菌均可扩增出6个基因(empA、vah1、vah4、flaA、rtxA和tonB)目的条带,未扩增出virA和angM基因;针对vah4和rtxA设计引物进行双重PCR扩增,同一PCR反应体系可扩增出两条目的条带,灵敏度为2.4×103 CFU/ml,对照菌无任何扩增条带;以vah4设计引物进行LAMP扩增,病原鳗弧菌可扩增出阶梯状条带,呈现阳性反应,6株对照菌无阶梯状扩增条带且呈现阴性反应,LAMP扩增灵敏度为2.4×101 CFU/ml。LAMP检测灵敏度是双重PCR的100倍,LAMP技术与PCR比较,操作简便、快速、灵敏度高且不需昂贵仪器,LAMP检测鳗弧菌的方法更适合于养殖生产实际应用。 相似文献
3.
《渔业科学进展》2015,(6)
本研究检测了分离自发病大菱鲆、半滑舌鳎及鲤鱼的22株病原鳗弧菌(Vibrio anguillarum)毒力相关基因的携带情况,并建立了病原鳗弧菌的分子生物学检测方法。以PCR方法检测8个毒力相关基因的分布,结果显示,22株病原鳗弧菌均可扩增出6个基因(empA、vah1、vah4、flaA、rtxA和tonB)目的条带,未扩增出virA和angM基因;针对vah4和rtxA设计引物进行双重PCR扩增,同一PCR反应体系可扩增出两条目的条带,灵敏度为2.4×103 CFU/ml,对照菌无任何扩增条带;以vah4设计引物进行LAMP扩增,病原鳗弧菌可扩增出阶梯状条带,呈现阳性反应,6株对照菌无阶梯状扩增条带且呈现阴性反应,LAMP扩增灵敏度为2.4×10~1 CFU/ml。LAMP检测灵敏度是双重PCR的100倍,LAMP技术与PCR比较,操作简便、快速、灵敏度高且不需昂贵仪器,LAMP检测鳗弧菌的方法更适合于养殖生产实际应用。 相似文献
4.
采用淋巴细胞杂交瘤技术制备抗豚鼠气单胞菌单克隆抗体分泌细胞株,获取抗豚鼠气单胞菌单克隆抗体,柠檬酸三钠还原氯金酸制备胶体金颗粒,选择直径20 nm的胶体金颗粒,标记抗豚鼠气单胞菌单克隆抗体并制备胶体金垫。将胶体金垫与喷涂有抗豚鼠气单胞菌兔多克隆抗体和羊抗鼠抗体的硝酸纤维素膜及样品吸收垫等组装成免疫层析试纸条,建立豚鼠气单胞菌的快速检测方法。用灭活细菌与血清混合的模拟样本测定试纸条的特异性、灵敏度,结果显示,试纸条对嗜水气单胞菌、温和气单胞菌、鳗弧菌、溶藻弧菌、荧光假单胞菌、副溶血弧菌等13种常见病原菌没有交叉反应,与豚鼠气单胞菌显示特异性反应,检测灵敏度为1×106 CFU/mL,结果显示时间小于5 min。研制的豚鼠气单胞菌胶体金免疫层析检测试纸条具有快速、简便、特异性高、适用于基层临床生产推广应用等优点。 相似文献
5.
病原迟钝爱德华菌毒力基因及双重PCR与LAMP检测方法的建立 总被引：2，自引：0，他引：2
为明确牙鲆及大菱鲆病原迟钝爱德华菌毒力基因的携带情况并建立分子检测方法,实验以迟钝爱德华菌fimA、fimB、gadB及citC为靶基因设计特异性引物,进行PCR扩增及环介导恒温扩增(loop-mediated isothermal amplification,LAMP),并对其特异性、灵敏性和实际应用进行了比较。结果显示,10株病原迟钝爱德华菌均扩增出fimA、fimB、gadB及citC 4种毒力基因,目的条带大小分别为240、217、171及119 bp,4株对照菌无任何扩增条带;以fimA和gadB设计的两对引物进行的双重PCR扩增,同一PCR反应体系中病原迟钝爱德华菌可扩增出240和171 bp两条目的条带,且灵敏度为3.0×103 CFU/mL,4株对照菌无任何扩增条带;以fimA设计的4条特异引物进行的LAMP扩增,病原迟钝爱德华菌可扩增出阶梯状条带,反应产物加入荧光染料SYBR Green Ⅰ后反应液呈现明显的绿色阳性反应,4株对照菌无阶梯状扩增条带且呈现橙色阴性反应,灵敏度为3.0×101 CFU/mL,比双重PCR的检测限要低100倍。研究表明,操作简便、快速且特异性及灵敏性强的LAMP检测方法,对迟钝爱德华菌引起的水产动物疾病的快速诊断具有实践意义。 相似文献
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从患病许氏平鲉的病灶处分离得到一株优势菌,记为SS-1。通过两点背部肌肉注射进行人工感染实验,证明该菌对健康许氏平鲉的半数致死浓度为9.6×106CFU/mL,可感染许氏平鲉的多种内脏和组织。细菌形态学和生理生化特征测定结果显示,菌株SS-1为革兰氏阴性,短杆状,极生单鞭毛;氧化酶阳性、接触酶阳性、硝酸盐还原阳性、吲哚产生阳性、V-P试验阳性、精氨酸双水解酶阳性、H2S产生阴性等,与鳗利斯顿氏菌特征相符。16S rRNA基因序列分析结果表明,该菌与鳗利斯顿氏菌的同源性达到了99%,因此,将菌株SS-1鉴定为鳗利斯顿氏菌。 相似文献
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本研究制备了鳗弧菌Vibrio anguillarum、杀鲑气单胞菌Aeromonas salmonicida、副溶血弧菌V. parahaemolyticus、哈维氏弧菌V. harveyi和腐败希瓦氏菌Shewanella putrefaciens 5株鲆鲽鱼类病原菌的兔抗血清,建立了5种菌的间接ELISA检测方法,并将该检测方法用于鱼类细菌分离物病原检测。人工感染结果显示,5株菌对大菱鲆的半数致死量(LD50)在102~107CFU/fish;制备的兔抗血清效价分别为1∶2 048 000、1∶16 000、1∶16 000、1∶1 024 000、1∶128 000;交叉反应结果显示,鳗弧菌、副溶血弧菌、哈维氏弧菌3株弧菌与抗血清相互之间存在交叉反应;抗血清特异检测灵敏度分别为104、108、107、105、106cell/ml;对13株海水鱼类细菌分离物进行检测,有1株腐败希瓦氏菌阳性,两株哈维氏弧菌阳性;1株副溶血弧菌和哈维氏弧菌均为阳性,该结果与16S rDNA序列的分子分析方法一致。 相似文献
8.
鲤病原鳗利斯顿氏菌的分离鉴定及生物学特性研究 总被引：1，自引：0，他引：1
从患病鲤(Cyprinus carpio L.)体内分离到一株优势生长菌,人工感染试验证明该菌对鲤有较强的致病性。对分离菌进行了形态特征、理化特性等生物学性状检验;测定了分离菌的16S rRNA和gyrB基因的部分序列,并与相关细菌16S rRNA和gyrB基因序列进行比对后,构建了基于两种基因的系统发生树。结果显示:分离菌所扩增的16S rRNA和gyrB基因序列与GenBank数据库中鳗利斯顿氏菌的16S rRNA和gyrB基因序列相似性均在97%以上,其16S rRNA基因序列长度为1449 bp(GenBank登录号:FJ824662),gyrB基因序列长度为1202 bp(Gen-Bank登录号:GQ452957);胞外酶活性及溶血活性检测表明分离菌具有淀粉酶、蛋白酶、明胶酶、卵磷脂酶活性,但不具有脂酶活性;在含7%家兔脱纤血液营养琼脂培养基上,呈β型溶血,分离菌均具有金属蛋白酶基因及溶血素基因。根据分离菌的表型特征及分子特征,判定分离菌为利斯顿氏菌属(Listonella MacDonell and Colwell1986)的鳗利斯顿氏菌(Listonella anguillarum)。分离菌的耐药谱测定结果显示,对供试49种抗菌药物中的青霉素G等12种药物耐药,对克林霉素等5种药物敏感,对恩诺沙星等32种药物高度敏感。 相似文献
9.
根据NCBI公布的爱德华氏菌(Edwardsiella ictaluri)磷酸丝氨酸转氨酶(phosphoserine transaminase,serC)的基因序列,设计一对特异性引物,建立了可快速而准确地检测爱德华氏菌的PCR方法,并对患病鱼组织进行了检测。研究结果表明,使用设计的引物能够扩增出与预计大小一致的124 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50 pg/反应,对靶标细菌的检测灵敏度为56 cfu/反应。样品的检测结果与实际发病情况一致,表明成功建立了爱德华氏菌常规PCR检测方法,可用于爱德华氏菌病的诊断。 相似文献
10.
根据ISAV的基因保守序列,利用LAMP Designer软件设计了6条引物,采用新型的环介导等温扩增设备进行扩增和检测,优化了反应条件,分析了所建立方法的特异性和灵敏度,并与RT-PCR和实时荧光RT-PCR进行比较。研究表明,该方法最适反应温度为64℃,反应10 min就可以观察到明显的扩增。该方法灵敏度高,检测限为78.4 fg RNA,比常规RT-PCR灵敏度高100倍,与实时荧光定量RT-PCR灵敏度相当;特异性好,与传染性胰腺坏死病毒(IPNV)、鲤春病毒血症病毒(SVCV)、出血性败血症病毒(VHSV)、鱼类病毒性神经坏死病病毒(VNNV)、鱼腹水病毒(YAV)等14种主要鱼类病毒没有交叉反应。结果表明,本研究建立了ISAV的实时荧光环介导等温扩增检测方法,实验能对整个扩增过程进行实时监测,提高检测灵敏度的同时,防止由于开盖跑电泳或加染料而导致的污染。 相似文献
11.
Rapid and sensitive detection of Vibrio harveyi by loop‐mediated isothermal amplification combined with lateral flow dipstick targeted to vhhP2 gene 
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下载免费PDF全文 Kanittada Thongkao Siwaporn Longyant Kun Silprasit Paisarn Sithigorngul Parin Chaivisuthangkura 《Aquaculture Research》2015,46(5):1122-1131
Vibrio harveyi is a causative agent of the Vibriosis or luminescent bacterial disease in worldwide aquaculture industry. A reliable assay for identification of V. harveyi infection is important to prevent the bacterial spread. In this study, biotinylated loop‐mediated isothermal amplification (LAMP) amplicons were produced by a set of four designed primers that recognized specifically the V. harveyi vhhP2 gene, encoding a putative outer membrane protein with unknown function, followed by hybridization with an fluorescein isothiocyanate (FITC)‐labelled probe and lateral flow dipstick (LFD) detection. A novel set of PCR primer was also designed specifically to vhhP2 gene and appear to be a species‐specific tool for V. harveyi detection. The optimized time and temperature conditions for the LAMP assay were 90 min at 65°C. The LAMP‐LFD and PCR methods accurately identified 22 isolates of V. harveyi but did not detect 16 non‐harveyi Vibrio isolates, and 34 non‐Vibrio bacterial isolates. The sensitivity of LAMP‐LFD for V. harveyi detection in pure culture was 1.1 × 102 CFU mL?1 or equivalent to 0.6 CFU per reaction, while that of PCR was 6 CFU per reaction. For spiked shrimp sample, the sensitivity of LAMP was 1.8 × 103 CFU g?1 or equivalent to 5 CFU per reaction, while that of PCR was 50 CFU per reaction. In conclusion, the established LAMP‐LFD methods provided a valuable tool for rapid identification of V. harveyi and can be used to distinguish V. harveyi from V. campbellii. 相似文献
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Rapid visual detection of cyprinid herpesvirus 2 by recombinase polymerase amplification combined with a lateral flow dipstick 下载免费PDF全文
下载免费PDF全文 Herpesviral haematopoietic necrosis (HVHN), caused by cyprinid herpesvirus 2 (CyHV‐2), causes significant losses in crucian carp (Carassius carassius) aquaculture. Rapid and convenient DNA assay detection of CyHV‐2 is useful for field diagnosis. Recombinase polymerase amplification (RPA) is a novel isothermal DNA amplification and detection technology that can amplify DNA within 30 min at ~37°C by simulating in vivo DNA recombination. Herein, a rapid and convenient detection assay based on RPA with a lateral flow dipstick (LFD) was developed for detecting CyHV‐2. The highly conserved ORF72 of CyHV‐2 was targeted by specific and sensitive primers and probes. The optimized assay takes only 15 min at 38°C using a water bath, with analysis of products by 2% agarose gel electrophoresis within 30 min. A simple lateral flow strip based on the unique probe in reaction buffer was developed for visualization. The entire RPA‐LFD assay takes 50 min less than the routine PCR method, is 100 times more sensitive and displays no cross‐reaction with other aquatic viruses. The combined isothermal RPA and lateral flow assay (RPA‐LFD) provides a simple, rapid, reliable method that could improve field diagnosis of CyHV‐2 when resources are limited. 相似文献
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Detection of koi herpesvirus in common carp, Cyprinus carpio L., by loop-mediated isothermal amplification 总被引：8，自引：0，他引：8
Loop-mediated isothermal amplification (LAMP) is a novel method that amplifies DNA with high specificity and rapidity under isothermal conditions. In this study, using the LAMP method, a protocol for koi herpes virus (KHV) detection in common carp was designed. A set of four primers, two inner and two outer, were designed based on the sequence of the thymidine kinase (tk) gene of KHV. Time and temperature conditions for detection of KHV were optimized for 60 min at 65 degrees C. The detection limit using LAMP was found to be similar to that by polymerase chain reaction. In this study, we have developed a highly sensitive and rapid diagnostic procedure for detection of KHV infection in common carp. 相似文献
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白斑综合征病毒环介导等温扩增快速检测方法的建立 总被引：1，自引：0，他引：1
根据对虾白斑综合征病毒(WSSV)囊膜蛋白VP28基因保守序列,利用Primer Explorerv 4.0软件设计了4条引物,建立了白斑综合征病毒环介导等温扩增快速检测方法,对反应温度和反应时间等参数进行了优化,同时将建立的LAMP检测方法与巢式PCR进行了比较分析。结果表明,LAMP最适反应在64℃恒温条件60min内完成,凝胶电泳呈现梯型条带;反应体系中添加SYBR Green I荧光染料后,绿色的阳性结果明显区别于橙色阴性结果。LAMP方法的最低检出限为100拷贝/μL,灵敏度较巢式PCR高100倍,而且LAMP方法在1h内即可完成检测,操作简单,无需复杂仪器,肉眼可直接观察检测结果。用建立的LAMP方法对临床发病南美白对虾样品进行了检测,结果表明,LAMP方法适合对虾白斑综合征病毒的现场快速检测。 相似文献
15.
A loop-mediated isothermal amplification (LAMP) procedure is described for rapid diagnosis of white tail disease, a viral disease caused by Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus (XSV), in the giant freshwater prawn, Macrobrachium rosenbergii. This method was more sensitive than conventional RT-PCR for detecting the two viruses. A set of four primers, two outer and two inner, were designed for MrNV detection. An additional pair of loop primers was also used in an accelerated LAMP reaction for detection of XSV. Time and temperature conditions were optimized for detection of the two viruses. The LAMP reaction is highly suited for disease diagnosis in developing countries as amplification of DNA can be detected without the use of agarose gel electrophoresis, by the production of whitish precipitate of magnesium pyrophosphate as a by-product. 相似文献
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Loop-mediated isothermal amplification (LAMP) is a novel technique for nucleic acid amplification with high specificity, sensitivity and rapidity and does not require expensive equipment or reagents. In the present study, we developed and evaluated a LAMP method for the rapid detection of Renibacterium salmoninarum causing the bacterial kidney disease in salmonids. This method was more sensitive than quantitative real-time polymerase chain reaction (qPCR). Using DNA template extracted from cultured R. salmoninarum , the LAMP method gave an amplification signal from template diluted to 10−8 while the limit of detection of qPCR was10−7 . The LAMP method was also highly specific and did not amplify DNA purified from five other Gram-positive and -negative bacterial fish pathogens. The method also worked well using extracts of macrophages infected with R. salmoninarum and kidney material from rainbow trout, which were positive for R. salmoninarum by qPCR and crude R. salmoninarum culture. There was some evidence for inhibitors of the LAMP reaction in the kidney samples, which was overcome by diluting the sample. 相似文献
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血卵涡鞭虫病是海水甲壳类的重要寄生虫病,其流行范围广、死亡率高、危害非常严重。本研究根据GenBank中已登录的血卵涡鞭虫(Hematodinium sp.)ITS1序列设计了1套引物,该引物可识别目标基因中6个不同区段。以此套引物建立了一种基于环介导恒温扩增技术(Loop-mediated isothermal amplification,LAMP)的血卵涡鞕虫病诊断方法。特异性试验结果表明,该套引物对血卵涡鞕虫检测具有较高的特异性,能有效检出血卵涡鞕虫。敏感度试验结果表明,该LAMP技术的灵敏度比常规PCR技术高4个数量级。分别运用LAMP和常规PCR技术对25份临床疑似病例进行检测,LAMP方法共检出感染病例25份,常规PCR检出感染病例23份。该技术能在65℃恒温条件下45～60min完成目的DNA的扩增,可直接通过肉眼观察反应产物中是否产生白色沉淀或经SYBRGreenI染色后通过颜色变化、扩增产物的琼脂糖凝胶电泳来定性判断结果,这将为血卵涡鞕虫病的临床诊断提供一种更加简便、快速、实用的方法。 相似文献