传染性造血器官坏死病毒环介导等温扩增检测方法的建立和初步应用
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摘要
实验建立了一套反转录环介导等温扩增方法(reverse transcription loop-mediated isothermal amplification,RT-LAMP),用于实验室及现场检测鲑鳟鱼传染性造血器官坏死病毒(infectious haematopoietic necrosis virus,IHNV)。针对IHNV的核蛋白基因设计特异性引物,以IHNV基因组RNA为模板,在反转录酶及Bst DNA聚合酶的作用下进行反转录LAMP,结果阳性样本表现为荧光绿色,阴性样本仍为红棕色。反应条件优化结果表明最适反应温度为64℃;特异性试验表明仅IHNV发生特异性扩增,而SVCV、HRV、VHSV和H_2O均不发生反应;敏感性试验表明该LAMP方法可检出浓度为1.0×10~(-4)μg/μL的IHNV核酸。本研究所建立的IHNV反转录LAMP检测法成本低、操作简单、反应迅速、不依赖专门的仪器,同时具有较高的灵敏度和特异性,适合IHNV的现场检测和大批量样品的检疫。
A reverse transcription loop-mediated isothermal amplification method for detection of trout infectious haematopoietic necrosis virus(IHNV) in both of laboratory and field was established in this study. A set of specific primers were designed based on the IHNV N gene sequence. IHNV RNA was used as a template for RT-LAMP in a reaction mixture containing reverse transcriptase and Bst DNA polymerase. Positive samples exhibited a fluorescent green color whereas negative samples exhibited a reddish brown color. The optimum temperature for the reaction was 64 ℃. Specific tests results showed that this method was capable to detect the IHNV had no cross action with SVCV, HRV, VHSV and H_2O. Sensitivity test results showed that this method could detect 1.0×10~(-4) μg/μL IHNV RNA. In summary, the method established in this study was suitable for field detection of IHNV and convenient perform quarantine of mass samples with high specificity and sensitivity, rapidity, simple, low cost, and without relying on any special equipment.
A reverse transcription loop-mediated isothermal amplification method for detection of trout infectious haematopoietic necrosis virus(IHNV) in both of laboratory and field was established in this study. A set of specific primers were designed based on the IHNV N gene sequence. IHNV RNA was used as a template for RT-LAMP in a reaction mixture containing reverse transcriptase and Bst DNA polymerase. Positive samples exhibited a fluorescent green color whereas negative samples exhibited a reddish brown color. The optimum temperature for the reaction was 64 ℃. Specific tests results showed that this method was capable to detect the IHNV had no cross action with SVCV, HRV, VHSV and H_2O. Sensitivity test results showed that this method could detect 1.0×10~(-4) μg/μL IHNV RNA. In summary, the method established in this study was suitable for field detection of IHNV and convenient perform quarantine of mass samples with high specificity and sensitivity, rapidity, simple, low cost, and without relying on any special equipment.
引文
[1]茆安婷,尹玉伟,代静,等.传染性造血器官坏死病毒研究进展[J].中国兽医杂志,2017,(10):79-82.
[2]孙红岩,刘文青,赵宝华.鱼类造血器官坏死病病毒检测及其防控研究进展[J].动物医学进展,2014,(5):90-94.
[3]余泽辉,耿毅,汪开毓,等.四川地区一株传染性造血器官坏死病毒的分离鉴定及系统发育分析[J].水产学报,2015,39(5):745-753.
[4]Ahmadivand S,Soltani M,Mardani K,et al.Infectious hematopoietic necrosis virus(IHNV) outbreak in farmed rainbow trout in Iran:Viral isolation,pathological findings,molecular confirmation,and genetic analysis [J].Virus Res,2017,229:17-23.
[5]Drolet B S,Chiou P P,Heidel J,et al.Detection of truncated virus particles in a persistent RNA virus infection in vivo [J].J Virol,1995,69(4):2140-2147.
[6]Soo J K,Eun Y L,Myung J O,et al.Comparison of IHNV detection limits by IMS-RT-PCR,western blot and ELISA [J].Fish Aquat Sci,2001,4(1):32-38.
[7]Phelps N B,Patnayak D P,Jiang Y,et al.The use of a one-step real-time reverse transcription polymerase chain reaction(rRT-PCR) for the surveillance of viral hemorrhagic septicemia virus(VHSV) in Minnesota [J].J Aquat Anim Health,2012,24(4):238-243.
[8]赵飞,邹为民.LAMP法在水产动物病原快速检测中的应用[J].南方水产科学,2007,3(2):71-75.
[9]刘淼,徐黎明,卢彤岩,等.鱼类传染性造血器官坏死病毒RT-LAMP检测方法的建立及应用[J].中国水产科学,2014,21(5):1065-1071.
[10]Gunimaladevi I,Kono T,Lapatra S E,et al.A loop mediated isothermal amplification(LAMP) method for detection of infectious hematopoietic necrosis virus(IHNV) in rainbow trout (Oncorhynchus mykiss) [J].Arch Virol,2005,150(5):899-909.
[11]Suebsing R,Kim J H,Kim S R,et al.Detection of viruses in farmed rainbow trout (Oncorhynchus mykiss) in Korea by RT-LAMP assay [J].J Microbiol,2011,49(5):741-746.
[12]何俊强,史秀杰,卢体康,等.传染性造血器官坏死病毒的新型环介导等温扩增(LAMP)荧光检测技术[J].中国动物检疫,2014,31(6):68-73.
[2]孙红岩,刘文青,赵宝华.鱼类造血器官坏死病病毒检测及其防控研究进展[J].动物医学进展,2014,(5):90-94.
[3]余泽辉,耿毅,汪开毓,等.四川地区一株传染性造血器官坏死病毒的分离鉴定及系统发育分析[J].水产学报,2015,39(5):745-753.
[4]Ahmadivand S,Soltani M,Mardani K,et al.Infectious hematopoietic necrosis virus(IHNV) outbreak in farmed rainbow trout in Iran:Viral isolation,pathological findings,molecular confirmation,and genetic analysis [J].Virus Res,2017,229:17-23.
[5]Drolet B S,Chiou P P,Heidel J,et al.Detection of truncated virus particles in a persistent RNA virus infection in vivo [J].J Virol,1995,69(4):2140-2147.
[6]Soo J K,Eun Y L,Myung J O,et al.Comparison of IHNV detection limits by IMS-RT-PCR,western blot and ELISA [J].Fish Aquat Sci,2001,4(1):32-38.
[7]Phelps N B,Patnayak D P,Jiang Y,et al.The use of a one-step real-time reverse transcription polymerase chain reaction(rRT-PCR) for the surveillance of viral hemorrhagic septicemia virus(VHSV) in Minnesota [J].J Aquat Anim Health,2012,24(4):238-243.
[8]赵飞,邹为民.LAMP法在水产动物病原快速检测中的应用[J].南方水产科学,2007,3(2):71-75.
[9]刘淼,徐黎明,卢彤岩,等.鱼类传染性造血器官坏死病毒RT-LAMP检测方法的建立及应用[J].中国水产科学,2014,21(5):1065-1071.
[10]Gunimaladevi I,Kono T,Lapatra S E,et al.A loop mediated isothermal amplification(LAMP) method for detection of infectious hematopoietic necrosis virus(IHNV) in rainbow trout (Oncorhynchus mykiss) [J].Arch Virol,2005,150(5):899-909.
[11]Suebsing R,Kim J H,Kim S R,et al.Detection of viruses in farmed rainbow trout (Oncorhynchus mykiss) in Korea by RT-LAMP assay [J].J Microbiol,2011,49(5):741-746.
[12]何俊强,史秀杰,卢体康,等.传染性造血器官坏死病毒的新型环介导等温扩增(LAMP)荧光检测技术[J].中国动物检疫,2014,31(6):68-73.