技术方法

鸡胚致死孤儿病毒和鸡减蛋综合症病毒多重PCR检测方法的建立及初步应用

王淑菁，付瑞，李晓波，王吉，卫礼，巩薇，岳秉飞，贺争鸣

(中国食品药品检定研究院实验动物资源研究所，国家实验动物质量检测中心，北京100050)

【摘要】目的建立鸡胚致死孤儿病毒(CELO)和鸡减蛋综合症病毒(EDS)的多重PCR检测方法并进行初步应用。方法参照GenBank提供的基因序列，设计了2对特异性引物分别扩增CELO长纤突蛋白和EDS六邻体蛋白的基因序列，建立检测CELO和EDS的多重PCR方法，考察该方法的特异性和敏感性，并使用该方法检测流感疫苗主种子批病毒是否存在外源性禽腺病毒的污染。结果该多重PCR方法成功扩增得到了两条特异性目的条带，并经测序验证。该方法特异性好，灵敏度显示核酸最低检测量可达10-4μg/mL。使用该方法检测12批次流感疫苗主种子批病毒，外源性禽腺病毒的检测结果均为阴性。结论成功建立鸡胚致死孤儿病毒和鸡减蛋综合症病毒的多重PCR检测方法，灵敏度高，特异性好。在流感疫苗主种子批病毒的外源性禽腺病毒的检测中具有很高的使用价值和应用前景。

【关键词】鸡胚致死孤儿病毒；鸡减蛋综合症病毒；禽腺病毒；多重PCR

[基金项目]实验动物质量检测关键技术研究(2013BAK11B01)；中检院中青年发展研究基金课题(2013NC2)。

[作者简介]王淑菁(1985-)，女，助理研究员，研究方向：实验动物病毒学。Email: wsj2008gogo@163.com。

[通讯作者]贺争鸣(1957-)，男，研究员，研究方向：微生物学和免疫学。Email：zhengminghe57@163.com。

【中图分类号】S852.65 R332【文献标识码】 A

doi:10.3969.j.issn.1671.7856. 2015.001.012

Detection of chicken embryo lethal orphan virus and egg drop

syndrome virus by multiplex polymerase chain reaction

WANG Shu-jing，FU Rui，LI Xiao-bo，WANG Ji，WEI Li，GONG Wei，YUE Bing-fei，HE Zheng-ming

(National Institute for Food and Drug Control，Institute for Laboratory Animal Resources,

National Center for Monitoring of Laboratory Animal Health，Beijing 100050，China)

Abstract【】ObjectiveTo establish multiplex PCR assay for detection of chicken embryo lethal orphan virus(CELO)and egg drop syndrome virus (EDS). Methods According to GenBank gene sequence, two pairs of specific primers designed were amplified CELO long fiber protein and EDS hexon protein gene sequence. The specificity and sensitivity of multiplex PCR were tested. We also use the multiplex PCR to detect exogenous CELO and EDS in influenza virus. ResultsTwo target bands have been successfully amplified and verified by sequencing. The specificity of the method is better, and the sensitivity is 10-4μg/mL. The results of detecting exogenous CELO and EDS in 12 influenza virus were negative. ConclusionThe multiplex PCR assay for detection of CELO and EDS was established successfully, which have good specificity and high sensitivity, and have high value and application prospect for detecting exogenous CELO and EDS in influenza virus.

【Key words】Chicken embryo lethal orphan virus(CELO)；Egg drop syndrome virus (EDS)；Avian adenovirus；Multiplex PCR

鸡胚致死孤儿病毒(CELO)属于禽腺病毒I型，为无包膜双股DNA病毒。……