A PCR Methodology for Detecting Eel Circovirus
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摘要: 鳗鲡圆环病毒（Eel circovirus，EeCV）是引起鳗鲡病毒性疾病的病原之一。根据GenBank已经发表的EeCV基因序列，设计一对特异性引物，对PCR反应条件优化，进行特异性、敏感性和重复性试验，建立了EeCV的PCR检测方法，并应用该方法对从福建省不同地区采集的160份疑似鳗鲡病毒性疾病病料进行了检测，其中65份表现为阳性，阳性率可达40.6%。结果表明，建立的PCR方法特异性强、敏感度高、重复性好，可以应用于EeCV引起的病害检测。
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关键词:
- 鳗鲡圆环病毒(EeCV) /
- PCR检测 /
- 方法建立 /
- 应用
Abstract: Eel circovirus (EeCV) is one of the pathogens that cause virosis on eels.Based on the gene sequence of EeCV in GenBank, a pair of primers was selected for the development of the detection method using PCR.The optimized reaction conditions, as well as the specificity, sensitivity and repeatability of the methodology were determined to establish the preliminary testing protocol. Subsequently, 160 epidemic specimens with the suspected viral infection were collected from are as in Fujian for the determination. Out of all samples, 65 were shown to be positive, at a rate of 40.6%. It appeared that the current PCR method could be employed to detect EeCV for early disease diagnosis on eels.-
Key words:
- eel circovirus /
- PCR detection /
- detection methodology /
- application
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图 1 PCR扩增结果
注：M为DL2000 DNA Marker，1为PCR扩增产物，2为Blank Control。
Figure 1. Result of PCR amplification
图 3 EeCV PCR检测方法特异性分析
注：M为DL2000 DNA Marker，1为EeCV，2为EHV，3为EIV，4为PCV，5为Blank Control。
Figure 3. Specificity of EeCV PCR detection method
图 4 EeCV PCR检测方法敏感性分析
注：M为DL2000 DNA Marker，1为阳性克隆质粒DNA(70 ng·μL-1)，2~10为阳性克隆质粒DNA5-1~5-9稀释液，11为Blank Control。
Figure 4. Sensitivity of EeCV PCR detection method
图 5 EeCV PCR检测方法重复性实验
注：M为DL2000 DNA Marker，1~3为样品XS20170223aa01检测结果，3~6为样品LY170512aa6检测结果，7~9为样品CL170602BM1aa6-1检测结果，10~11为样品HZ20170218nm1-1检测结果，13~15为样品CL170321ar02检测结果，16~18为样品CLbm170531ar2L检测结果，19.Blank Control。
Figure 5. Repeatability of EeCV PCR detection method
表 1 160份疑似鳗鲡病毒性疾病病料EeCV检测结果
Table 1. EeCV detection results on 160 epidemic specimens with suspected eel virosis
采集地区 鳗鲡品种 检测部位 样品数 阳性样品数 阳性率/% 龙岩 美洲鳗鲡 鳃、肝、肾、脾 6 1 16.7 漳平 美洲鳗鲡 鳃、肝、肾、脾 21 1 4.8 建瓯 欧洲鳗鲡 鳃、肝、肾、脾 5 4 80.0 建阳 美洲鳗鲡 鳃、肝、肾、脾 11 8 72.7 霞浦 美洲鳗鲡 鳃、肝、肾、脾 24 10 41.7 长乐 美洲鳗鲡 鳃、肝、肾、脾 56 22 39.3 永泰 美洲鳗鲡、欧洲鳗鲡 鳃、肝、肾、脾 13 8 61.5 清流 美洲鳗鲡 鳃、肝、肾、脾 8 1 12.5 罗源 欧洲鳗鲡 鳃、肝、肾、脾 6 4 66.7 南安 欧洲鳗鲡 鳃、肝、肾、脾 10 6 60.0 合计 160 65 40.6 
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