Establishment and application of duplex PCR detection system between Anguillid herpesvirus and Eel circovirus
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摘要:
目的 建立能同时检测鳗鲡疱疹病毒（Anguillid herpesvirus，AHV）与鳗鲡圆环病毒（Eel circovirus，EeCV）的双重PCR法。 方法 优选PCR引物组合，建立鳗鲡疱疹病毒与鳗鲡圆环病毒的双重PCR检测方法，探究双重PCR扩增条件，并检验该方法的特异性、灵敏性和临床样品检测效果。 结果 双重PCR法同时扩增出鳗鲡疱疹病毒690 bp和鳗鲡圆环病毒338 bp特异性片段，与猪圆环病毒（Porcine circovirus）、鸭圆环病毒（Duck circovirus）、锦鲤疱疹病毒（Koi herpesvirus）和鲤疱疹病毒（Cyprinid herpesvirus）的核酸不发生扩增反应。鳗鲡疱疹病毒和鳗鲡圆环病毒重组质粒标准品的最低全检出量为5.0×10-4 μg·mL-1。用所建立的双重PCR方法对50份临床病鳗进行检验，其中鳗鲡疱疹病毒的感染率为16%，鳗鲡圆环病毒的感染率为68%，共感染率为8%。该感染率与单一PCR检测结果完全一致。 结论 建立的鳗鲡疱疹病毒和鳗鲡圆环病毒双重PCR检测法不仅特异性强、灵敏度高、重复性好，而且临床应用效果佳，为鳗鲡疱疹病毒和鳗鲡圆环病毒的快速检测提供了有效的技术支持。 Abstract:Objective The aim of the present study was to establish a duplex PCR method for detection of Anguillid herpesvirus and Eel circovirus. Method Routine PCR primers were preferred to optimize the amplification conditions and test the specificity and sensitivity of the method. At the same time, the sick eel of suspected cases collected from different farm were detected by the duplex PCR method, and compared with the routine PCR method. Result The results showed that the specific fragments of 690 bp and 388 bp were amplified by using the duplex PCR from DNA samples of Anguillid herpesvirus and Eel circovirus, respectively. But the nucleic acid amplification results of Porcine circovirus, Duck circovirus, Koi herpesvirus and Cyprinid herpesvirus were negative. When the positive DNA concentrations of both Anguillid herpesvirus and Eel circovirus were diluted to 5.0×10-4 μg·mL-1, the corresponding target fragments could still be detected. 50 samples collected from suspected cases of sick eels were diagnosed by PCR. The infection rate of Anguillid herpesvirus was 16%. The infection rate of Eel circovirus was 68%. The infection rate of Anguillid herpesvirus and Eel circovirus was 8%. Conclusion The results indicate that the duplex PCR detection method has certain specificity and sensitivity, and can be used for the detection and differential diagnosis of recessive cases of clinical infection of Anguillid herpesvirus and Eel circovirus． -
Key words:
- Anguillid herpesvirus /
- Eel circovirus /
- duplex PCR /
- test method
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图 1 单一PCR扩增结果
1：阴性对照；2：AHV；3：EeCV；4：阴性对照；M：DL-2000 Marker。
Figure 1. Routine PCR amplification results
1: Negative control; 2: AHV; 3: EeCV; 4: Negative control; M:DL-2000 Marker.
图 2 双重PCR退火温度优化
1：阴性对照；2～9表示温度为50、50.9、52.3、54、56.3、58.1、59.3、60 ℃；M：DL-2000 Marker。
Figure 2. Duplex PCR annealing temperature optimization
1: Negative control; 2–9: 50, 50.9, 52.3, 54, 56.3, 58.1, 59.3, 60 ℃; M: DL-2000 Marker.
图 3 双重PCR引物浓度优化
1：阴性对照；2–6：引物浓度分别为4、6、8、10、12 μmol·L−1；M：DL-2000 Marker。
Figure 3. Duplex PCR primer concentration optimization
1: Negative control; 2–6: 4, 6, 8, 10, 12 μmol·L−1 of primer concentration; M: DL-2000 Marker.
图 4 双重PCR特异性检验结果
1：猪圆环病毒；2：鸭圆环病毒；3：鲤疱疹病毒；4：锦鲤疱疹病毒；5：阴性对照；M：DL-2000 Marker。
Figure 4. Results of the duplex PCR specificity test
1: Porcine circovirus; 2: Duck circovirus; 3: Cyprinid herpesvirus; 4: Koi herpesvirus; 5: Negative control; M: DL-2000 Marker.
图 5 双重PCR敏感性检测结果
M：DL-2000 Marker；1：阴性对照；2–10：扩增浓度5.0×10−8、5.0×10−7 、5.0×10−6、5.0×10−5、5.0×10−4 、5.0×10−3、5.0×10−2、5.0×10−1、5.0×100 μg·mL−1。
Figure 5. Results of duplex PCR sensitivity detection
M: DL-2000 Marker; 1:Negative control; 2–10: 5.0×10−8, 5.0×10−7, 5.0×10−6, 5.0×10−5, 5.0×10−4, 5.0×10−3, 5.0×10−2 , 5.0×10−1, 5.0×100 μg·mL−1.
图 6 双重PCR临床样品检验结果
a：AHV和EeCV双重PCR结果；b：AHV单一PCR结果；c：EeCV单一PCR结果。1～6：临床样；7：阴性对照；M：DL-2000 Marker。
Figure 6. Results of the duplex PCR clinical sample test
a:The duplex PCR results for AHV and EeCV; b: Routine PCR result for AHV; c:Routine PCR results of EeCV. 1–6: Clinical samples; 7: Negative control; M:DL-2000 Marker.
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