PCV2a、PCV2b和PCV2d多重PCR检测方法的建立及应用
Establishment and application of multiplex PCR assay for PCV2a,PCV2b and PCV2d
谭健梅 1赵雨欣 1黄艳玲 1周小汇 1陈媛 2雷磊 1罗施乐 2郝裕波 1杜红梅 3雷红宇 1苏建明 1王乃东 1湛洋1
作者信息
- 1. 湖南农业大学动物医学院,湖南长沙 410128;兽用蛋白质工程疫苗湖南省重点实验室,湖南长沙 410128
- 2. 湖南派智生物科技有限公司,湖南长沙 410205
- 3. 常德市畜牧水产事务中心,湖南常德 415000
- 折叠
摘要
为了建立一种能够快速鉴别PCV2a、PCV2b和PCV2d的方法,本研究针对PCV2a、PCV2b和PCV2d这3个基因型分别设计3组特异性引物,通过构建阳性标准质粒,筛选出最适引物组合,建立了一种PCV2分型的多重PCR检测方法,并对该方法进行优化后,开展特异性、灵敏度、重复性试验以及临床样品检测.结果显示,利用筛选出的最适引物组合建立了多重PCR检测方法,能特异性扩增出3个目的条带:PCV2a(321 bp)、PCV2b(190 bp)和PCV2d(561 bp),探索出最佳退火温度和循环数分别为60 ℃和25个,与其他病原(PPV、PRV、PEDV、TGEV、PRRSV、CSFV)无交叉反应;对构建的阳性标准质粒的最低检测拷贝数为103 copies;同一模板的3次重复性试验结果一致;检测2个猪场流行的PCV2基因型毒株,并通过测序和进化分析进一步验证了该检测方法的准确性.结果表明,本研究建立的多重PCR检测方法有助于开展PCV2a、PCV2b和PCV2d的快速鉴别诊断,为PCV2的防控以及流行病学研究提供了技术支持.
Abstract
The purpose of this study was to develop a rapid detection method for identification of PCV2a,PCV2b and PCV2d,three sets of specific primers were designed for the three genotypes of PCV2a,PCV2b and PCV2d,respectively.The optimized sets of primers were selected by constructed positive standard plasmids,and a multiplex PCR method for identification of PCV2 genotypes was established.The reaction conditions were optimized and the specificity experiments,sensitivity experiments and re-peatability experiments were verified,and clinical samples were used for preliminary application by this method.The results showed that a multiplex PCR detection method was established by using the opti-mal sets of primers,which could specifically amplify three target fragments:PCV2a(321 bp),PCV2b(190 bp)and PCV2d(561 bp).Furthermore,the optimum annealing temperature was 60 ℃ and cycle number was 25.This method had no cross-reaction with other pathogens(PPV,PRV,PEDV,TGEV,PRRSV,CSFV),and the limit of detection was 103 copies by using the positive standard plasmid.Besides,this method had good repeatability with coefficients of three repeatability tests using the same template.The PCV2 strains prevalent in 2 pig farms were detected by the method,the main genotype was determined,and the accuracy of the detection method was further verified by sequencing and evolutionary analysis.The multiplex PCR detection method developed in this study was conducive to the rapid identified diagnosis of PCV2a,PCV2b and PCV2d,and provided technical support for the control of PCV2 and epidemiological studies.
关键词
PCV2a/PCV2b/PCV2d/共感染/多重PCRKey words
PCV2a/PCV2b/PCV2d/co-infection/multiple PCR引用本文复制引用
基金项目
国家自然科学基金项目(32202790)
湖南省自然科学基金项目(2022JJ40183)
湖南省技术攻关"揭榜挂帅"项目(2021NK1030)
湖南省青年科技人才项目(2022RC1046)
湖南省教育厅科学研究重点项目(23A0194)
出版年
2024
国家科技期刊平台
NSTL
万方数据