猪主要繁殖障碍疾病病原的多重实时定量PCR快速检测

畜牧兽医学报

• 研究简报 • 上一篇

猪主要繁殖障碍疾病病原的多重实时定量PCR快速检测

赵绪永，赵丽，宁豫昌，马辉

（郑州牧业工程高等专科学校，郑州 450011）

收稿日期:2012-06-01 出版日期:2012-12-26 发布日期:2012-12-26
作者简介:赵绪永 (1973-), 男, 河南新野人, 博士, 主要从事动物疫病分子病原学研究， E-mail：Zhaoxy6868@163.com
基金资助:
河南省教育厅自然科学研究计划项目(2011A230016)；河南省重点科技攻关项目（092102110073）

Simultaneous Detection of Major Pathogens of Reproductive Failure Diseases in Swine by a Multiplex Real-time PCR

ZHAO Xu-yong, ZHAO Li, NING Yu-chang, MA Hui

(Zhengzhou College of Animal Husbandry Engineering, Zhengzhou 450011, China)

Received:2012-06-01 Online:2012-12-26 Published:2012-12-26

摘要/Abstract

摘要：

本研究旨在建立多重Real-time PCR方法检测猪繁殖与呼吸综合征病毒(PRRSV)、猪圆环病毒2型（PCV2）、猪伪狂犬病毒（PRV）和猪细小病毒(PPV)。根据GenBank数据库中PRRSV、PCV2、PRV、和PPV的核苷酸序列设计特异性引物和探针，通过优化反应条件，检测系列稀释的纯化阳性质粒，建立检测PRRSV、PCV2、PRV和PPV的单项和多重Real-time PCR方法。灵敏度和特异性试验结果表明，无论是单项和多重Real-time PCR 检测，该方法都具有高度特异性，与其它病原检测无明显交叉反应；对4种病毒的最低检测量为＜10¹拷贝或1 TCID₅₀，检测灵敏度比常规PCR高100倍。对40份临床样本进行检测，多重Real-time PCR检测结果和单项Real-time PCR检测结果完全一致。建立的同时检测PRRSV、PCV2、PRV和PPV的多重Real-time PCR方法具有快速、灵敏、特异、重复性好和能对样品进行定量检测等优点。

Abstract:

The aim of this study is to establish a multiplex real-time PCR assay to simultaneously detect and discriminate porcine reproductive and respiratory syndrome virus (PRRSV)，porcine circovirus type 2 (PCV2)，porcine pseudorabies virus（PRV）and porcine parvovirus (PPV) in a single test tube. According to the nucleotide sequences of PRRSV，PCV2, PRV and PPV from GenBank, four pairs of specific probes were designed. By optimizing the probe’s concentration, primers concentration and sample DNA extraction, a multiplex real-time quantitative PCR assay for detection of these viruses was established. The specificity and sensitivity of the multiplex real-time quantitative PCR assay were analyzed for single virus and multiple viruses. The results showed that the specificity was high, and no amplification was achieved to the other pathogeneses. Both multiplex and singleplex assays were consistently able to detect ＜10¹ copies of plasmid templates or 1 TCID₅₀ virus per reaction. 40 clinical samples were comparatively detected using the multiplex and singleplex assay. The data showed that the results of the singleplex and multiplex assay were in accordance with the results of conventional PCR. A multiplex real-time PCR assay was developed to simultaneously detect and discriminate PRRSV, PCV2, PRV and PPV in a single test tube, the method is rapid, sensitive, specific and accurate.

中图分类号:

S852.285.3

赵绪永，赵丽，宁豫昌，马辉. 猪主要繁殖障碍疾病病原的多重实时定量PCR快速检测[J]. 畜牧兽医学报, doi: .

ZHAO Xu-yong, ZHAO Li, NING Yu-chang, MA Hui. Simultaneous Detection of Major Pathogens of Reproductive Failure Diseases in Swine by a Multiplex Real-time PCR[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, doi: .

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.xmsyxb.com/CN/

https://www.xmsyxb.com/CN/Y2012/V43/I12/1998

参考文献

［1］王泽洲，于勇，吴越，等. 猪生殖障碍疾病的研究概况［J］. 中国兽医科技, 2007, 37 (9) :823-826.

［2］殷震, 刘景华. 动物病毒学［M］. 第2版. 北京: 科学出版社, 1997: 538-548.

［3］STRAW B E, ALLAIRE S D, WILLIAM L M . 猪病学［M］. 第8版. 北京:中国农业大学出版社, 2003: 189-253.

［4］HARMS P A, HALBUR P G, SORDEN S D. Three cases of porcine respiratory disease complex associated with porcine circovirus type 2 infection［J］. Swine Health Prod, 2002,10(1): 27-30.

［5］KIM J, CHOI C, CHAE C. Pathogenesis of postweaning multisystemic wasting syndrome reproduced by co-infection with Korean solates of porcine circovirus 2 and porcine parvovirus［J］. J Comp Pathol,2003,128(1):52-59.

［6］许立华, 王玲, 芦银华，等.三种猪繁殖障碍性病毒混合感染的分子生物学调查［J］. 中国兽医科技, 2004, 34(7) : 40-43.

［7］蒋大良, 余兴龙, 李润成，等. 猪瘟病毒NS3 基因克隆、原核表达及间接 ELISA 方法初步建立［J］. 微生物学通报, 2010, 37(1): 78-84.

［8］丛晓燕, 吴家强, 李俊，等.重组N 蛋白抗原检测美洲型 PRRSV 抗体间接ELI SA 方法的建立［J］. 中国动物检疫, 2010, 27(12): 40-44.

［9］李素, 李尚波, 王文成，等. 阻断ELISA与中和试验检测猪瘟疫苗免疫猪血清抗体的比较［J］. 动物医学进展,2007, 28(10): 40-43.

［10］范晓娟, 李刚, 史利军，等. 检测猪圆环病毒2型抗体SPA胶体金免疫层析方法的建立［J］. 中国畜牧兽医, 2009, 36(7):158-161.

［11］杨宗照，母安雄，方维焕. 高致病性猪繁殖与呼吸综合征病毒实时荧光定量PCR方法的建立［J］. 中国预防兽医学报, 2009, 31(04): 288-291.

［12］黄律, 龙进学, 翟少伦，等. 猪细小病毒4型Taq Man 荧光定量PCR方法的建立与应用［J］. 动物医学进展, 2011, 32(1) : 1-9.

［13］赵丽, 崔保安, 陈红英，等. 实时荧光定量PCR检测伪狂犬病病毒方法的建立与初步应用［J］. 中国兽医学报, 2009,29(4)：433-436.

［14］刘俊平, 庄金山, 孙志，等. 检测猪圆环病毒2型TaqMan荧光定量PCR 方法的建立［J］. 上海交通大学学报( 农业科学版), 2007, 25(5): 483-488.

［15］STUERWALD N, COHEN J, HERRERA R J, et al. Analysis of gene expression in single oocytes and embryos by real-time rapid cycle fluorescence monitored RT-PCR［J］. Mol Hum Reprod, 1999, 5(11): 1034-1039.

［16］牛伟, 丁壮, 王晓莉，等. 实时荧光定量PCR方法检测猪繁殖与呼吸综合征病毒和猪瘟病毒［J］. 中国兽医学报, 2011,31(2):174-177.

［17］魏文康，吕殿红，温肖会，等. 食源性动物组织中CSFV和PRRSV双重双色荧光定量RT- PCR 检测方法的建立及应用［J］. 中国预防兽医学报, 2011, 33(08): 620-624.

［18］刘秀清, 宁金友, 赵荣茂，等. 西宁市猪圆环病毒2型感染的流行病学调查［J］. 中国兽医科学, 2006, 36( 2) : 162-165.

猪主要繁殖障碍疾病病原的多重实时定量PCR快速检测

Simultaneous Detection of Major Pathogens of Reproductive Failure Diseases in Swine by a Multiplex Real-time PCR

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 0

编辑推荐

Metrics

本文评价