鸡传染性贫血病毒荧光定量PCR检测方法的建立及其在疫苗污染检测中的应用

doi:10.11843/j.issn.0366-6964.2016.07.020

畜牧兽医学报 ›› 2016, Vol. 47 ›› Issue (7): 1459-1464.doi: 10.11843/j.issn.0366-6964.2016.07.020

鸡传染性贫血病毒荧光定量PCR检测方法的建立及其在疫苗污染检测中的应用

任志浩^#，房立春^#，栾怀彪，李阳，王一新，崔治中，常爽^*，赵鹏^*

（山东农业大学动物医学院，泰安 271018）

收稿日期:2016-04-10 出版日期:2016-07-23 发布日期:2016-07-21
通讯作者: 赵鹏，E-mail：zhaopeng@sdau.edu.cn;常爽，E-mail：changshuang81@126.com。赵鹏、常爽并列通信作者
作者简介:任志浩(1992-)，男，山东青岛人，硕士生，主要从事动物分子病毒学研究，E-mail：zhren92@163.com；房立春(1991-),男，山东济南人，硕士生，主要从事动物分子病毒学研究，E-mail：18854881003@163.com。两人对本研究有同等贡献，为并列第一作者
基金资助:
农业部财政项目“禽垂直传播性疾病诊断技术研究”

Development and Application of a Quantitative PCR for Detection of Chicken Infectious Anemia Virus Contamination in Attenuated Vaccines

REN Zhi-hao^#，FANG Li-chun^#，LUAN Huai-biao，LI Yang，WANG Yi-xin，CUI Zhi-zhong，CHANG Shuang^* ，ZHAO Peng^*

（College of Veterinary Medicine，Shandong Agricultural University，Tai’an 271018，China）

Received:2016-04-10 Online:2016-07-23 Published:2016-07-21

摘要/Abstract

摘要：

为了更快速而灵敏地检测禽弱毒疫苗中鸡传染性贫血病毒（CIAV）的污染，根据CIAV基因组保守序列设计合成了引物及TaqMan探针，通过优化反应条件建立了快速检测CIAV的实时荧光定量PCR检测方法。结果显示，建立的检测方法灵敏度可达10拷贝•μL－1，比常规PCR灵敏度高100倍。该方法与禽白血病病毒、禽网状内皮组织增生症病毒以及马立克病病毒等病毒基因均无交叉反应，具有很好的特异性；批内重复和批间重复显示变异系数均小于2%，呈现良好的可重复性。在模拟试验中，该方法能够检测到1 000羽份弱毒疫苗中1个EID50的低剂量污染，应用该方法从送检的39种（批）商品化禽用弱毒疫苗中检测到2份为CIAV阳性。上述结果表明，本研究建立的荧光定量PCR检测方法灵敏度高，特异性强，重复性好，可用于检测疫苗中CIAV低剂量的污染。

关键词: 鸡传染性贫血病毒, 实时荧光定量PCR, 弱毒疫苗, 污染

Abstract:

To detect the contamination of Chicken infectious anemia virus (CIAV) in attenuated vaccines for poultry，primers and TaqMan probes were designed and synthesized based on the published CIAV genome sequence，and a real-time quantitative PCR method for detecting the CIAV was established by optimizing the reaction conditions.Results analysis showed that the sensitivity of the qPCR may increase detection to 10 copies•μL－1，and the method was approximately 100-fold higher than the sensitivity of the conventional PCR.This detection method had good specificity，and showed no cross-reactions with ALV，REV and MDV genes，the CV (coefficient of variation) of intra-assay and inter-assay were less than 2%，and also showed that the method had the characteristics of better repeatability.In our experiment，a low dosage of 1 EID50•1 000－1 plumes and in 2 positive (5.1%) of 39 samples were detected for CIAV detection from commercial poultry vaccines by qPCR method.In this study，suggesting that this method provides a high sensitivity，strong specificity and better repeatability system for detection of a low dosage CIAV contamination in attenuated vaccines.

Key words: chicken infectious anemia virus, real-time PCR, live vaccine, contamination

中图分类号:

S852.659.2

任志浩，房立春，栾怀彪，李阳，王一新，崔治中，常爽，赵鹏. 鸡传染性贫血病毒荧光定量PCR检测方法的建立及其在疫苗污染检测中的应用[J]. 畜牧兽医学报, 2016, 47(7): 1459-1464.

REN Zhi-hao，FANG Li-chun，LUAN Huai-biao，LI Yang，WANG Yi-xin，CUI Zhi-zhong，CHANG Shuang，ZHAO Peng. Development and Application of a Quantitative PCR for Detection of Chicken Infectious Anemia Virus Contamination in Attenuated Vaccines[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2016, 47(7): 1459-1464.

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鸡传染性贫血病毒荧光定量PCR检测方法的建立及其在疫苗污染检测中的应用

Development and Application of a Quantitative PCR for Detection of Chicken Infectious Anemia Virus Contamination in Attenuated Vaccines

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