Rapid PCR Method for Differentiating Virulent and Vaccine Enteritis Viruses in Ducks
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摘要: 为建立鸭肠炎病毒（duck enteritis virus，DEV）强毒和疫苗弱毒的鉴别诊断方法，通过分析比较GenBank数据库中上传的DEV强毒株和疫苗弱毒株UL2基因核苷酸序列，分析DEV疫苗弱毒和DEV强毒在UL2基因上的核苷酸序列，利用引物设计软件Oligo 7.0，设计一组可对DEV强毒株和疫苗弱毒株UL2基因进行编码区全长扩增的特异性引物，经条件优化后建立DEV强毒和疫苗弱毒鉴别诊断的PCR方法。结果表明，DEV疫苗弱毒和DEV强毒在UL2基因上存在528 bp的连续核苷酸序列缺失。优化后的PCR方法最佳退火温度为55℃，对DEV强毒和疫苗弱毒扩增片段大小分别为1 019 bp和491 bp；敏感性强，最低检测限为15.3 pg；特异性好，对鸭源常见传染病（如番鸭细小病毒、鸭圆环病毒、鹅细小病毒、鸭源大肠杆菌、鸭疫里默氏杆菌和鸭源禽多杀性巴氏杆菌）均无特异性扩增。Abstract: To accurately and rapidly differentiate between the virulent and the attenuated vaccine strains of duck enteritis viruses (DEVs), sequences of the virus UL2 genes were retrieved from the databank at GenBank for the study. A 528 bp continuous nucleotide deletion region was found on the genes of both strains. Using the primer design software, Oligo 7.0, specific primers targeting for the development and optimization of the PCR differentiation diagnosis methodology based on the UL2 characterization were selected. The optimal annealing temperature was determined to be 55℃ rendering amplified segments for the DEV virulent and the vaccine strains at 1 019 bp and 491 bp, respectively. The established protocol was sensitive with a low detection limit of 15.3 pg; and, specific without cross-amplification with common duck origin pathogens, such as, Muscovy duck parvovirus, duck circovirus, goose parvovirus, Escherichia coli, Rimerella anatipstifer and Pasteurella multocida. Consequently, it was considered applicable for future studies on the pathogenic mechanism of DEV.
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Key words:
- duck enteritis virus /
- virulent strain /
- attenuated vaccine strain /
- UL2 gene /
- PCR /
- differential diagnosis
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图 2 PCR扩增和特异性试验
注：M为DL2000分子量标准；1为DEV疫苗弱毒株；2为DEV强毒；3为MDPV；4为DuCV；5为GPV；6为E. coli；7为R.A.；8为P.M.。
Figure 2. PCR amplification and specificity tests
图 3 PCR敏感性试验
注：M为DL2000分子量标准；1为1.53×104 pg；2为1.53×103pg；3为1.53×102 pg；4为1.53×101 pg；5为1.53×100 pg；6为阴性对照。
Figure 3. PCR sensitivity test
表 1 DEV不同毒力毒株UL2基因序列信息
Table 1. Information on UL2 gene of virulent and vaccine DEVs
毒株 GenBank号 长度/bp 毒力 地区 参考文献 Yulin/2016/60D KX925440 1002 强毒株virulent China - Yulin/2016/30D KX925439 1002 China - CV(CSC) KJ549663 1002 China Yang，et al.(2015)[12] CHv JQ647509 1002 China Wu，et al.(2012)[13] 2085 JF999965 1002 Germany Wang，et al.(2011)[14] N1 JQ043216 1002 China - N2 JQ248596 1002 China - N3 JQ248597 1002 China - LS JQ248598 1002 China - LH2011 KC480262 1002 China - Chinese commercial DEV vaccine EF449516 474 疫苗弱毒株vaccine China Li，et al.(2009)[15] VAC EU082088 477 China Li，et al.(2009)[16] attenuated strain 1 JQ347517 474 China - attenuated strain 2 JQ347518 474 China - C-KCE KF263690 474 China - K KF487736 474 China Yang，et al.(2015)[12] 注：-表示GenBank中未标注相关信息。 
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