猫泛白细胞减少症病毒环介导等温扩增检测方法的建立
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摘要
目的建立一种快速、简便检测猫泛白细胞减少症病毒(FPV)的环介导等温扩增(LAMP方法。方法针对Genbank中FPV基因组设计了4条LAMP扩增引物,通过优化反应温度和反应时间,确定高效的LAMP扩增条件。结果建立的LAMP方法在65℃水浴条件下反应60 min可对FPV进行高效扩增,检测限量为5.01×10~2拷贝/μL,比常规PCR检测的敏感性高10倍,且与猫疱疹病毒1型(FHV-1),犬细小病毒(CPV)及犬腺病毒(CAV)均无交叉反应。结论建立的LAMP方法设备要求低、特异性好、灵敏度高,适用于FPV临床样品的快速检测。
Objective To establish a rapid, convenient detection method of feline panleukopenia virus(FPV) by loop-mediated isothermal amplification(LAMP). Methods Two pairs of primers were designed according to the conserved sequences of FPV in Genbank. The efficient LAMP amplification conditions were determined by optimizing temperature and time. Results The results showed a highly efficient amplification for FPV nucleic acid which performed at 65 ℃for 60 min. It also showed a high sensibility with a detection limit of 5.01 × 10~2 copies/μL, 10 times higher than the conventional PCR detection in the sensitivity. There was no cross reaction with feline herpesvirus type 1(FHV-1), canine parvovirus(CPV) and vanine adenovirus(CAV). Conclusion The established LAMP method is good in duplication, stability, specificity, and sensitivity. This method can be used for the fast detection of FPV nucleic acid in clinical samples from cats.
Objective To establish a rapid, convenient detection method of feline panleukopenia virus(FPV) by loop-mediated isothermal amplification(LAMP). Methods Two pairs of primers were designed according to the conserved sequences of FPV in Genbank. The efficient LAMP amplification conditions were determined by optimizing temperature and time. Results The results showed a highly efficient amplification for FPV nucleic acid which performed at 65 ℃for 60 min. It also showed a high sensibility with a detection limit of 5.01 × 10~2 copies/μL, 10 times higher than the conventional PCR detection in the sensitivity. There was no cross reaction with feline herpesvirus type 1(FHV-1), canine parvovirus(CPV) and vanine adenovirus(CAV). Conclusion The established LAMP method is good in duplication, stability, specificity, and sensitivity. This method can be used for the fast detection of FPV nucleic acid in clinical samples from cats.
引文
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[8]张跃伟,李旭妮,郭盼盼,等.荧光显色在环介导等温扩增(LAMP)检测猪繁殖与呼吸综合征病毒的应用[J].农业生物技术学报,2010,18(3):508-513.
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[10]时建立,彭喆,王莉莉,等.猪流行性腹泻病毒LAMP检测方法的建立及初步应用[J].中国动物检疫,2016,33(7):82-85+89.
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[12]Nagamine K,Hase T,Notomi T.Accelerated reaction by loop-mediated isothermal amplication using loop primers[J].Mol Cell Probes,2002,16(3):223-229.
[13]罗亚坤,梁琳,王静,等.猪流行性腹泻病毒环介导等温扩增检测方法的建立及应用[J].中国畜牧兽医,2017,44(02):344-349.
[14]王翀,刘大飞,刘春国,等.同时检测猫细小病毒、杯状病毒、疱疹病毒1型多重PCR方法的建立[J].中国预防兽医学报,2014,36(01):31-33.
[15]邱杰,温肖会,翟少伦,等.猫泛白细胞减少症病毒VP2基因快速检测方法的建立与应用[J].中国动物传染病学报,2015,23(02):21-25.
[16]王吉,付瑞,卫礼,等.猫细小病毒PCR检测方法的建立及初步应用[J].实验动物科学,2015,32(1):1-6.
[2]An DJ,Jeong WJ,Jeong HYJ,et al.Phylogenetic analysis of feline panleukopenia virus(FPLV)strains in Korean cats[J].Res Vet Sci,2011,90(1):l63-l67.
[3]亢文华,赵凤龙,郝霖雨,等.猫泛白细胞减少症病毒的研究进展[J].中国畜牧兽医,2008,35(9):108-111.
[4]蔡宝祥.家畜传染病学[M].北京:中国农业出版社,2001,361-363.
[5]Notomi T,Okayama H,Masubuchi H,et al.Loop-mediated isothermal amplification of DNA[J].Nucleic Acids Res,2000,28(12):E63.
[6]Sun WW,Sun Q,Yan LP,et al.The application of S6110-baced loop-mediated isothermal amplification(LAMP)in the early diagnosis of tuberculous meningitis[J].Oncotarget,2017,8(34):57537-57542.
[7]包红梅.禽流感病毒RT-LAMP系列检测方法的建立和应用[D].哈尔滨:东北农业大学,2014.
[8]张跃伟,李旭妮,郭盼盼,等.荧光显色在环介导等温扩增(LAMP)检测猪繁殖与呼吸综合征病毒的应用[J].农业生物技术学报,2010,18(3):508-513.
[9]刘业兵,张磊,宁宜宝,等.猪细小病毒LAMP检测方法的建立[J].中国农业科学,2010,43(14):3012-3018.
[10]时建立,彭喆,王莉莉,等.猪流行性腹泻病毒LAMP检测方法的建立及初步应用[J].中国动物检疫,2016,33(7):82-85+89.
[11]于晶.猫泛白细胞减少症[J].中国畜牧兽医文摘,2016,32(11):212-212.
[12]Nagamine K,Hase T,Notomi T.Accelerated reaction by loop-mediated isothermal amplication using loop primers[J].Mol Cell Probes,2002,16(3):223-229.
[13]罗亚坤,梁琳,王静,等.猪流行性腹泻病毒环介导等温扩增检测方法的建立及应用[J].中国畜牧兽医,2017,44(02):344-349.
[14]王翀,刘大飞,刘春国,等.同时检测猫细小病毒、杯状病毒、疱疹病毒1型多重PCR方法的建立[J].中国预防兽医学报,2014,36(01):31-33.
[15]邱杰,温肖会,翟少伦,等.猫泛白细胞减少症病毒VP2基因快速检测方法的建立与应用[J].中国动物传染病学报,2015,23(02):21-25.
[16]王吉,付瑞,卫礼,等.猫细小病毒PCR检测方法的建立及初步应用[J].实验动物科学,2015,32(1):1-6.