鲤浮肿病毒荧光RAA快速检测方法的建立
|
摘要
为建立一种简单、快速的鲤浮肿病毒(CEV)重组酶介导的链替换扩增(RAA)检测方法,本研究通过比较分析CEV P4a基因序列,设计引物与探针,经过筛选优化,建立了实时荧光RAA检测方法。结果显示,该方法在39℃恒温反应20 min即可快速、特异性地检测出CEV,与常见的鱼类病毒不发生交叉反应,其对质粒标准品的检测下限为2.8×10~1拷贝/μL,组内和组间变异系数均小于5%。利用本研究建立的方法对35份临床鲤科鱼类样品进行检测,检测结果与荧光定量PCR方法结果一致。本研究建立的检测方法操作简单,特异性强、敏感性高,结果可靠,不依赖于复杂的仪器,适用于现场对CEV的快速检测。
To establish a rapid and simple method for the detection of carp edema virus(CEV), a real-time recombinase-aid amplification(RAA) assay was developed with the primers and probe designed according to the conserved sequence of CEV P4a gene. Through screening the primers and optimizing experimental conditions, the assay can be completed within 20 min at 39℃.The assay was specific to amplify the target sequence of CEV with a detection limit up to 2.8 ×10~1 copies per reaction. The co-efficient of variations in intra-and inter-assay were both less than 5%. In addition, 35 clinical samples were tested by this method and 7 samples were detected to be positive, which was 100% consistent with the real-time PCR. The real-time RAA assay developed in this study was a simple, rapid, sensitive, reliable and affordable method which could potentially be applied for the detection of CEV in the research laboratory and on site diagnosis.
To establish a rapid and simple method for the detection of carp edema virus(CEV), a real-time recombinase-aid amplification(RAA) assay was developed with the primers and probe designed according to the conserved sequence of CEV P4a gene. Through screening the primers and optimizing experimental conditions, the assay can be completed within 20 min at 39℃.The assay was specific to amplify the target sequence of CEV with a detection limit up to 2.8 ×10~1 copies per reaction. The co-efficient of variations in intra-and inter-assay were both less than 5%. In addition, 35 clinical samples were tested by this method and 7 samples were detected to be positive, which was 100% consistent with the real-time PCR. The real-time RAA assay developed in this study was a simple, rapid, sensitive, reliable and affordable method which could potentially be applied for the detection of CEV in the research laboratory and on site diagnosis.
引文
[1]Matras M,Borzym E,Stone D,et al.Carp edema virus in Polish aquaculture-evidence of significant sequence divergence and a new lineage in common carp Cyprinus carpio(L.)[J].JFish Dis,2016,40(3):319-325.
[2]Lewisch E,Gorgoglione B,Way K,et al.Carp edema virus/Koi sleepy disease:an emerging disease in Central-East Europe[J].Transbound Emerg Dis,2014,62(1):6-12.
[3]Miyazaki T,Isshiki T,Katsuyuki H.Histopathological and electron microscopy studies on sleepy disease of koi Cyprinus carpio koi in Japan[J].Dis Aquat Organ,2005,65(3):197-207.
[4]徐立蒲,王静波,张文,等.鲤鱼浮肿病流行情况的初步研究[J].检验检疫学刊,2016,26(05):14-16.
[5]徐立蒲,王小亮,李清,等.我国北方地区鲤浮肿病毒的流行情况调查与监测分析[J].水生动物学报,2018,42(05):935-941.
[6]Zhang Xiao-dong,Ni Ying-ying,Ye Jian,et al.Carp edema virus,an emerging threat to the carp(Cyprinus carpio)industry in China[J].Aquaculture,2017,474(1):34-39.
[7]温智清,刘莹,唐绍林,等.云南锦鲤养殖场鲤浮肿病毒的鉴定及基因型分析[J].病毒学报,2017,33(06):905-913.
[8]Oyamatsu T,Matoyama H,Yamamoto K,et al.A trial for the detection of carp edema virus by using polymerase chain reaction[J].Suisanzoshoku,1997,45:247-252.
[9]Adamek M,Jung-Schroers V,Hellmann J,et al.Concentration of carp edema virus(CEV)DNA in koi tissues affected by koi sleepy disease(KSD)[J].Dis Aquat Organ,2016,119(3):245-251.
[10]王小亮,徐立蒲,王姝,等.养殖鲤和锦鲤中的鲤浮肿病毒检测与分析[J].中国畜牧兽医,2017,44(10):3084-3089.
[11]吕蓓,程海荣,严庆丰,等.用重组酶介导扩增技术快速扩增核酸[J].中国科学:生命科学,2010,40(10):983-988.
[12]吕蓓,程海荣,严庆丰,等.体外核酸快速扩增技术的发展和不断创新[J].中国生物工程杂志,2011,31(3):91-96.
[13]Piepenburg O,Williams CH,Stemple D L,et al.DNA detection using recombination proteins[J].PLoS Biol,2006,4(7):1115-1121.
[2]Lewisch E,Gorgoglione B,Way K,et al.Carp edema virus/Koi sleepy disease:an emerging disease in Central-East Europe[J].Transbound Emerg Dis,2014,62(1):6-12.
[3]Miyazaki T,Isshiki T,Katsuyuki H.Histopathological and electron microscopy studies on sleepy disease of koi Cyprinus carpio koi in Japan[J].Dis Aquat Organ,2005,65(3):197-207.
[4]徐立蒲,王静波,张文,等.鲤鱼浮肿病流行情况的初步研究[J].检验检疫学刊,2016,26(05):14-16.
[5]徐立蒲,王小亮,李清,等.我国北方地区鲤浮肿病毒的流行情况调查与监测分析[J].水生动物学报,2018,42(05):935-941.
[6]Zhang Xiao-dong,Ni Ying-ying,Ye Jian,et al.Carp edema virus,an emerging threat to the carp(Cyprinus carpio)industry in China[J].Aquaculture,2017,474(1):34-39.
[7]温智清,刘莹,唐绍林,等.云南锦鲤养殖场鲤浮肿病毒的鉴定及基因型分析[J].病毒学报,2017,33(06):905-913.
[8]Oyamatsu T,Matoyama H,Yamamoto K,et al.A trial for the detection of carp edema virus by using polymerase chain reaction[J].Suisanzoshoku,1997,45:247-252.
[9]Adamek M,Jung-Schroers V,Hellmann J,et al.Concentration of carp edema virus(CEV)DNA in koi tissues affected by koi sleepy disease(KSD)[J].Dis Aquat Organ,2016,119(3):245-251.
[10]王小亮,徐立蒲,王姝,等.养殖鲤和锦鲤中的鲤浮肿病毒检测与分析[J].中国畜牧兽医,2017,44(10):3084-3089.
[11]吕蓓,程海荣,严庆丰,等.用重组酶介导扩增技术快速扩增核酸[J].中国科学:生命科学,2010,40(10):983-988.
[12]吕蓓,程海荣,严庆丰,等.体外核酸快速扩增技术的发展和不断创新[J].中国生物工程杂志,2011,31(3):91-96.
[13]Piepenburg O,Williams CH,Stemple D L,et al.DNA detection using recombination proteins[J].PLoS Biol,2006,4(7):1115-1121.