快速检测鱼类淋巴囊肿病毒和海洋双RNA病毒环介导等温扩增检测方法的建立与应用
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摘要
淋巴囊肿病毒(Lymphocystis disease virus,LCDV)广泛分布于世界各地的淡水、半咸水和海水中,可感染9目34科共计140种以上鱼类,是对鱼类危害较为严重的病毒病之一,其感染率可高达80%,死亡率可达30%。本研究根据GenBank登录的淋巴囊肿病毒的主要衣壳蛋白(MCP)基因序列(AB212999),选择其高度保守区域,利用Primer Explorer V3软件进行引物设计,建立了用于检测淋巴囊肿病毒的环介导恒温扩增方法,对LCDV-LAMP反应条件进行了优化,评估了该方法的特异性、灵敏度并用建立的方法对109尾牙鲆进行了检测。特异性试验证实,该方法只能检测LCDV核酸,与流行性造血器官坏死病毒、虎纹蛙病毒、蛙虹彩病毒、新加坡石斑虹彩病毒、鳜鱼传染性脾肾坏死病毒、真鲷虹彩病毒及对虾白斑综合症病毒都无交叉反应,具有高度的特异性。将LAMP与常规PCR及实时定量PCR的检测限进行比较分析,结果表明LAMP与实时定量PCR的检测限一致约为15龟,比常规PCR灵敏度高10倍。用建立的LCDV-LAMP与常规PCR检测109尾牙鲆样品,结果显示,两者对健康和发病严重的样品检测结果一致,但对无临床症状的样品存在8尾差异;经2次实时定量PCR复检差异样品,均与LAMP一致,表明LAMP较常规PCR的检测灵敏度更高,而且从样品的制备到获得结果仅需2 h,具有检测周期短的优势。以上结果表明,采用LAMP法检测淋巴囊肿病毒具有特异性好、灵敏度高、检测耗时短等特点,比较适合养殖场、出入境口岸进行淋巴囊肿病毒的现场、即时检测。
海洋双RNA病毒(Marine birnavirus,MABV)广泛分布于世界各地的海水中,可感染30科11种软体、4种甲壳动物和10多种鱼类,特别是对鲫幼鱼危害严重的病毒病之一,其感染率可高达85%,死亡率可达62%。本研究根据GenBank提供的海洋双RNA病毒聚合蛋白(polyprotein)的VP2基因序列(D61384),利用PrimerExplorer V3软件进行引物设计,建立了海洋双RNA病毒的反转录环介导恒温扩增方法。对MABV RT-LAMP反应条件进行了优化,评估了该方法的特异性和灵敏度,并采用优化了的方法对养殖场送检的鲈鱼、真鲷、大菱鲆和牙鲆各30尾进行MABV的检测。结果显示,该方法对MABV核酸有高度的特异性,与染性胰脏坏死病毒、传染性造血器官坏死病毒、鲤春血症病毒、病毒性出血败血症病毒及病毒性神经坏死病毒都无交叉反应。灵敏性试验发现,该方法最低检测限为16.6fg总RNA,与实时定量RT-PCR的检测限一致,但比常规PCR灵敏度高10倍。对养殖场送检的120份样品进行RT-LAMP和实时定量PCR海洋双RNA病毒检测,结果显示两种方法的检测结果完全一致,表明RT-LAMP具有更高的灵敏度。以上结果表明,本研究所建立的MABV RT-LAMP能对海洋双RNA病毒进行准确、快速的检测,具有特异性好、灵敏度高、检测成本低廉等优点,可用于基层实验室和出入境检疫对海洋双RNA病毒属的疫情监测。
Lymphocystis disease virus (LCDV) is distributed widely in worldwide freshwater, brackish water and seawater, and can infect more than 140 species of fishes belonging to 9 orders 34 families which is one of the harmful virus diseases to fish. Its infection rate was up to 80 % and the mortality rate could reach 30 %. A specific and sensitive method of a loop-mediated isothermal amplification (LAMP) was developed for rapid detection of lymphocystis disease virus (LCDV). The specific primers were designed according to the highly conservative sequence of major capsid protein (MCP) gene of LCDV using Primer Explorer V3 software. The primers and the reaction condition were optimized to LAMP assy. Moreover, the specificity and sensitivity were estimated and 109 Paralichthys olivaceus samples were tested with this method. It was found that there was high specificity and no cross-reaction with EHNV, TFV, BIV, SGIV, ISKNV, RSIV and WSSV. The detection limit of LAMP assay was 15 fg and similar to real-time quantitative PCR, but 10-fold higher than conventional PCR. The LAMP assay was evaluated using 109 clinical samples and there were the same results between healthy and seriously infected samples, but had 8 differences in unkown samples. The results of 2 detection times indicated real-time quantitative PCR and LAMP were uniform, more sensitive than conventional PCR. Moreover, the results were obtained from extraction of viral DNA using this method only 2 hours. Above all, the LAMP method can effectively detect LCDV at fish farms and in entry-exit inspection and quarantine, and it is credible for specificity, sensitivity and rapidity.
Marine birnavirus is existed widely in worldwide seawater and could infect more than 11 species of mollusc, 4 shellfish and more than 10 fishes, which belong to 30 families. It was specialy harmful to young of seriola quinqueradiata and its infection rate was up to 85 %, the mortality rate could reach 62 %. A reverse transcription loop-mediated isothermal amplification (RT-LAMP) method was developed for marine birnavirus. According to sequence of polyprotein (VP2) gene of MABV from Genbank the specific primers were designed using Primer Explorer V3 software. The RT-LAMP was optimized for its primers and reaction condition. Furthermore, the specificity and sensitinity of RT-LAMP were estimated and Lateolabrax japonicus, Pagrosomus major, Scophthalmus maximus, Paralichthys olivaceus from farms were tested 30 respectively. This assay had high specificity and no cross-reaction among IPNV, IHNV, SVCV, VHSV and VNNV. The detection limit of RT-LAMP was 16.6 fg total RNA and similar to real-time reverse transcription quantitative PCR, but 10-fold higher than conventional reverse transcription PCR. The results of 120 fish samples demonstrated RT-LAMP and real-time quantitative RT-PCR both were good sensitivity. Above results shown RT-LAMP was exact, rapid, specific, sensitive, low-cost assay for MABV, and could be used for fish farms and in entry-exit inspection and quarantine for early diagnosis of MABV.
海洋双RNA病毒(Marine birnavirus,MABV)广泛分布于世界各地的海水中,可感染30科11种软体、4种甲壳动物和10多种鱼类,特别是对鲫幼鱼危害严重的病毒病之一,其感染率可高达85%,死亡率可达62%。本研究根据GenBank提供的海洋双RNA病毒聚合蛋白(polyprotein)的VP2基因序列(D61384),利用PrimerExplorer V3软件进行引物设计,建立了海洋双RNA病毒的反转录环介导恒温扩增方法。对MABV RT-LAMP反应条件进行了优化,评估了该方法的特异性和灵敏度,并采用优化了的方法对养殖场送检的鲈鱼、真鲷、大菱鲆和牙鲆各30尾进行MABV的检测。结果显示,该方法对MABV核酸有高度的特异性,与染性胰脏坏死病毒、传染性造血器官坏死病毒、鲤春血症病毒、病毒性出血败血症病毒及病毒性神经坏死病毒都无交叉反应。灵敏性试验发现,该方法最低检测限为16.6fg总RNA,与实时定量RT-PCR的检测限一致,但比常规PCR灵敏度高10倍。对养殖场送检的120份样品进行RT-LAMP和实时定量PCR海洋双RNA病毒检测,结果显示两种方法的检测结果完全一致,表明RT-LAMP具有更高的灵敏度。以上结果表明,本研究所建立的MABV RT-LAMP能对海洋双RNA病毒进行准确、快速的检测,具有特异性好、灵敏度高、检测成本低廉等优点,可用于基层实验室和出入境检疫对海洋双RNA病毒属的疫情监测。
Lymphocystis disease virus (LCDV) is distributed widely in worldwide freshwater, brackish water and seawater, and can infect more than 140 species of fishes belonging to 9 orders 34 families which is one of the harmful virus diseases to fish. Its infection rate was up to 80 % and the mortality rate could reach 30 %. A specific and sensitive method of a loop-mediated isothermal amplification (LAMP) was developed for rapid detection of lymphocystis disease virus (LCDV). The specific primers were designed according to the highly conservative sequence of major capsid protein (MCP) gene of LCDV using Primer Explorer V3 software. The primers and the reaction condition were optimized to LAMP assy. Moreover, the specificity and sensitivity were estimated and 109 Paralichthys olivaceus samples were tested with this method. It was found that there was high specificity and no cross-reaction with EHNV, TFV, BIV, SGIV, ISKNV, RSIV and WSSV. The detection limit of LAMP assay was 15 fg and similar to real-time quantitative PCR, but 10-fold higher than conventional PCR. The LAMP assay was evaluated using 109 clinical samples and there were the same results between healthy and seriously infected samples, but had 8 differences in unkown samples. The results of 2 detection times indicated real-time quantitative PCR and LAMP were uniform, more sensitive than conventional PCR. Moreover, the results were obtained from extraction of viral DNA using this method only 2 hours. Above all, the LAMP method can effectively detect LCDV at fish farms and in entry-exit inspection and quarantine, and it is credible for specificity, sensitivity and rapidity.
Marine birnavirus is existed widely in worldwide seawater and could infect more than 11 species of mollusc, 4 shellfish and more than 10 fishes, which belong to 30 families. It was specialy harmful to young of seriola quinqueradiata and its infection rate was up to 85 %, the mortality rate could reach 62 %. A reverse transcription loop-mediated isothermal amplification (RT-LAMP) method was developed for marine birnavirus. According to sequence of polyprotein (VP2) gene of MABV from Genbank the specific primers were designed using Primer Explorer V3 software. The RT-LAMP was optimized for its primers and reaction condition. Furthermore, the specificity and sensitinity of RT-LAMP were estimated and Lateolabrax japonicus, Pagrosomus major, Scophthalmus maximus, Paralichthys olivaceus from farms were tested 30 respectively. This assay had high specificity and no cross-reaction among IPNV, IHNV, SVCV, VHSV and VNNV. The detection limit of RT-LAMP was 16.6 fg total RNA and similar to real-time reverse transcription quantitative PCR, but 10-fold higher than conventional reverse transcription PCR. The results of 120 fish samples demonstrated RT-LAMP and real-time quantitative RT-PCR both were good sensitivity. Above results shown RT-LAMP was exact, rapid, specific, sensitive, low-cost assay for MABV, and could be used for fish farms and in entry-exit inspection and quarantine for early diagnosis of MABV.
引文
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