猪流行性腹泻病毒SYBR Green Ⅰ实时荧光定量PCR检测方法的建立
Development of SYBR Green Ⅰ Real-Time PCR for Detection of Porcine Epidemic Diarrhea Virus
董苏洁 1孔宁 2秦文珍 2翟焕杰 2翟雪滢 2杨心雨 2童武 2郑浩 2于海 2童光志 2李有文 3单同领2
作者信息
- 1. 塔里木大学动物科学与技术学院,阿拉尔 843300;中国农业科学院上海兽医研究所,上海 200241
- 2. 中国农业科学院上海兽医研究所,上海 200241
- 3. 塔里木大学动物科学与技术学院,阿拉尔 843300
- 折叠
摘要
为了建立高效、灵敏的猪流行性腹泻病毒(PEDV)检测方法,本研究从GenBank数据库中获取PEDV N基因序列,扩增出PEDVN基因标准质粒,并在N基因的保守区域内设计了一对特异性荧光定量引物,成功建立了 SYBR Green Ⅰ实时荧光定量PCR检测方法.经过一系列试验表明,该检测方法线性关系良好,R2值为0.99;特异性强,敏感性高,最低可检测至2.23 copies/μL,比普通PCR灵敏约100倍;重复性好,组内变异系数为0.25％～0.43％,组间变异系数为0.67％～0.97％;对于各地区96份临床样品检测出PEDV阳性率为25％.本研究建立的实时荧光定量PCR检测方法为PEDV的临床诊断、流行病学调查以及定量研究提供了有效的检测工具.
Abstract
In order to develop an efficient and sensitive detection method for Porcine epidemic diarrhea virus(PEDV),the PEDV N gene sequence was obtained from GenBank database and a pair of specific primers were designed from its conservative region for development of a SYBR Green Ⅰ real-time PCR method.A series of experiments were performed for optimization and the optimal factors included the linear correlation coefficient at 0.99 and detection limit at 2.23 copies/μL,which was about 100 times more sensitive than conventional PCR.The coefficient of variation within groups was between 0.25％-0.43％and between groups was between 0.67％-0.97％,indicating its good repeatability.Total 96 clinical samples from various regions were tested using this method and the PEDV positive rate was 25％(24/96).The real-time PCR method developed in this study provided an effective tool for clinical diagnosis,epidemiological investigation and quantitative research of PEDV.
关键词
猪流行性腹泻病毒/N基因/SYBR/Green/Ⅰ/荧光定量PCRKey words
Porcine epidemic diarrhea virus/N gene/SYBR Green Ⅰ/real-time PCR引用本文复制引用
基金项目
国家自然科学基金(31872478)
国家自然科学基金(32102665)
国家自然科学基金(31760741)
上海市自然科学基金(19ZR1469100)
出版年
2024
国家科技期刊平台
NSTL
万方数据