[1]郭瑶,汪平,董沁芳,等.同时检测猪圆环病毒2型、猪细小病毒和伪狂犬病毒连接酶检测反应-PCR基因芯片检测方法的建立[J].中国预防兽医学报,2011,(07):526-530.
 GUO Yao,WANG Ping,DONG Qin-fang,et al.A universal oligonucleotide microarray based on ligase detection reaction (LDR)-PCR for parallel detection of porcine circovirus type 2, porcine parvovirus and pseudorabies virus[J].Chinese journal of preventive veterinary medicine,2011,(07):526-530.
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同时检测猪圆环病毒2型、猪细小病毒和伪狂犬病毒连接酶检测反应-PCR基因芯片检测方法的建立
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《中国预防兽医学报》[ISSN:1008-0589/CN:23-1417/S]

卷:
期数:
2011年07期
页码:
526-530
栏目:
诊断和检测技术
出版日期:
2011-07-15

文章信息/Info

Title:
A universal oligonucleotide microarray based on ligase detection reaction (LDR)-PCR for parallel detection of porcine circovirus type 2, porcine parvovirus and pseudorabies virus
作者:
郭瑶;汪平;董沁芳;程菊会;徐辉;丁先锋;郭江峰;姜永厚;
浙江理工大学生命科学学院;浙江省动物疫病预防控制中心;
Author(s):
GUO Yao1 WANG Ping1 DONG Qin-fang1 CHENG Ju-hui1 XU Hui2 DING Xian-feng1 GUO Jiang-feng1 JIANG Yong-hou1
1. College of Life Sciences, Zhejiang Sci-Tech University, Hangzhou 310018, China; 2. Centre of Animal Diesase Control of Zhejiang Province, Hangzhou 310020, China
关键词:
LDR-PCR通用芯片多重检测猪病毒
Keywords:
LDR-PCR universal microarray parallel detection swine viruses
摘要:
为快速、灵敏、准确地同时检测和鉴别猪圆环病毒2型(PCV2)、猪细小病毒(PPV)和伪狂犬病毒(PRV)的方法,本研究采用连接酶检测反应(LDR)-PCR和基因芯片技术建立一种新型检测方法。首先在3种病毒的保守区内分别设计一对LDR探针,两端各连接一段通用序列,依次进行LDR、通用引物荧光标记扩增和芯片杂交,同时比较引物标记和Cy5-dCTP标记方法的灵敏度。结果表明该方法可以特异地检测PCV2、PPV和PRV3种病毒,而对牛病毒性腹泻病毒、猪传染性胃肠炎病毒、猪流行性腹泻病毒、猪繁殖与呼吸障碍综合征病毒、猪瘟病毒、乙型脑炎病毒、猪圆环病毒1型检测结果均为阴性;对3种病毒的最低检测限少于10个拷贝;Cy5-dCTP标记检测的灵敏度显著高于引物标记。利用建立的方法对41例临床样品进行检测,与普通PCR检测结果符合率为97.6%~100%。该方法的建立为基础研究和临床应用提供了技术平台。
Abstract:
To establish a sensitive detection method for porcine circovirus type 2 (PCV2), porcine parvovirus (PPV) and pseudorabies virus (PRV) which cause swine severe reproductive and/or respiratory failure, a novel diagnostic oligonucleotide microarray based on ligase detection reaction PCR (LDR-PCR) was developed in this study. According to alignment of the viral sequences, LDR probes for each virus were designed, which were flanked by universal sequences on both sides of the probes. Through asymmetric PCR enrichment of the ligation products by universal primers, labeling and microarray hybridization, the target viruses were detected and differentiated. Compared to the labeling method with Cy5-primer, Cy5-dCTP labeling was more sensitive in this assay. The specific test result showed that the assay had no cross reaction with bovine viral diarrhea (BVDV), transmissible gastroenteritis virus (TGEV), porcine epidemic diarrhea virus (PEDV), porcine reproductive and respiratory syndrome virus (PRRSV), classical swine fever virus (CSFV), Japanese encephalitis virus (JEV) and PCV1. The sensitivity of the assay was able to detect as low as 10 copies of target virus. Forty one clinical samples were tested by the assay and traditional PCR, the results of both methods showed a concordance rate of 97.6% to 100%. The established assay provided a new technical platform for the basic research and clinical application

参考文献/References:

[1] 姜永厚,徐辉,商晗武,朱良俊,陈伟杰,赵灵燕,方立. 多重PCR同时检测猪圆环病毒2型,猪细小病毒,猪繁殖与呼吸综合征病毒和猪瘟病毒[J]. 中国兽医学报. 2009年10期
[2] 张海燕,马文丽,李凌,吴清华,郑文岭. 应用不对称PCR技术提高寡核苷酸基因芯片杂交效率[J]. 军医进修学院学报. 2005年04期

备注/Memo

备注/Memo:
基金:浙江省科技计划项目(2008C22081);;浙江省自然科学基金(Y3090166);;浙江省科技攻关重点项目(2005C22032);;浙江省生物医学重中之重学科开放基金(SWYX0908)
更新日期/Last Update: 2011-09-29