Establishment of One-step RT-PCR for Batai Virus Detection in Ducks
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摘要: 为建立鸭巴泰病毒（Batai virus，BATV）的快速检测方法，本研究基于该病毒囊膜蛋白（M）基因序列的保守区域，经优化反应条件，对检测方法的特异性、敏感性及重复性进行了测定，建立了一种用于检测BATV的一步法RT-PCR快速诊断方法。所建立的检测方法可特异性扩增出BATV M基因480 bp的序列片段，其最低检测病毒量为1×101.1 TCID50，且重复性好，对禽坦布苏病毒、鸭甲肝病毒等5种鸭常见传染病病原均未扩增出相应的片段，特异性好。以上结果表明，所建立的RT-PCR检测方法具有很好的特异性和敏感性，可用于鸭BATV感染的早期快速诊断及流行病学调查。Abstract: For a rapid detection of Batai virus (BATV) in ducks, a one-step reverse transcription polymerase chain reaction (RT-PCR) assay was developed based on the conserved region of the M gene in BATV. With the optimized reaction conditions, this assay could specifically amplify a 480 bp fragment from the M gene. There were no similar gene amplifications on 5 other pathogens found in ducks, such as avian Tembusu virus and duck hepatitis virus. The testing sensitivity of the method was 1×101.1 TCID50 with a high reproducibility. Consequently, this newly developed methodology seemed to be highly specific and sensitive for BATV detection, and could be applied for rapid and early diagnosis as well as epidemiological investigations on BATV.
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Key words:
- batai virus /
- duck /
- RT-PCR
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图 1 不同退火温度的RT-PCR扩增效果
注：M为DL2000，1为46.0℃，2为48℃，3为50.3℃，4为52.4℃，5为54.8℃，6为56.6℃，7为58.2℃，8为62℃，9为阴性对照。
Figure 1. Amplifications of RT-PCR with different anneal temperatures
图 2 不同引物浓度的RT-PCR扩增效果
注：M为DL2000，1为阴性对照，2为0.04 μmol·L-1，3为0.1 μmol·L-1，4为0.2 μmol·L-1，5为0.4 μmol·L-1，6为0.8 μmol·L-1。
Figure 2. Amplifications of RT-PCR with different primer concentrations
图 3 RT-PCR检测方法特异性
注：M为DL2000，1为禽坦布苏病毒，2为BATV，3为禽1型副黏病毒，4为H9亚型禽流感病毒，5为鸭甲肝病毒，6为鸭呼肠孤病毒，7为阴性对照。
Figure 3. Specificity of One-step RT-PCR assay
图 4 RT-PCR检测敏感性
注：M为DL2000，1~7为10-0-10-6不同稀释度的BATV溶液。
Figure 4. Sensitivity of One-step RT-PCR assay
图 5 临床样品检测的部分电泳结果
注：M为DL2000，1为阴性对照，2~10为临床样品。
Figure 5. Detection results of partial clinical samples
表 1 扩增序列与已有毒株序列的同源性
Table 1. Homology of sequence between amplified fragment and others in GenBank
毒株名 序列登录号 同源性/% ZJ2014 KU746870.1 100 XQ-B KJ398937.1 99 NM/12 KJ187039.1 99 Chittoor/IG-20217 JX846599.1 96 MM2222 JX846596.1 96 ON-1/E/94 AB257764.1 96 
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表 2 14份组织样品的重复检测结果
Table 2. Results on 14 samples with duplicate detections by One-step RT-PCR assay
组织样品 第1次检测阳性样品数/样品总数 第2次检测阳性样品数/样品总数 肝脏 5/5 5/5 脾脏 5/5 5/5 肾脏 4/4 4/4
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